Recombinant Simian virus 41 Hemagglutinin-neuraminidase (HN)

What is the Simian virus 41 hemagglutinin-neuraminidase (HN) protein and what are its primary functions?

The Simian virus 41 (SV41) hemagglutinin-neuraminidase (HN) is a multifunctional surface glycoprotein found in parainfluenza viruses. It serves three primary functions: (1) receptor recognition and binding to sialic acid-containing receptors on host cells; (2) neuraminidase activity that cleaves sialic acid to prevent viral self-aggregation and facilitate viral release; and (3) promotion of fusion activity through interaction with the viral fusion (F) protein. This HN-F protein interaction is virus type-specific and constitutes a prerequisite for mediating both virus-cell fusion and cell-cell fusion processes in most parainfluenza viruses. The molecular basis of this functional interaction remains partially obscure, particularly regarding which regions of the F protein are responsible for physical interaction with the HN protein .

How does SV41 HN compare structurally and functionally with other paramyxovirus HN proteins?

SV41 HN shares structural similarities with other paramyxovirus HN proteins, yet maintains distinctive characteristics that determine its specificity. Comparative analyses have shown that while SV41 HN can functionally interact with its cognate F protein, it displays selective compatibility with F proteins from other viruses. For instance, research has demonstrated that the SV41 F protein can induce cell fusion when coexpressed with the HN protein of human parainfluenza virus type 2 (PIV-2), but the PIV-2 F protein cannot induce fusion with the SV41 HN protein . Additionally, unlike the PIV-2 F protein which induces extensive cell fusion with mumps virus (MuV) HN protein, the SV41 F protein does not . These functional differences highlight the unique structural properties of SV41 HN that govern its selective interactions with F proteins across different paramyxoviruses.

What are the recommended cell lines and expression systems for studying recombinant SV41 HN protein?

For optimal expression and functional studies of recombinant SV41 HN protein, several cell lines and expression systems have proven effective. Human cervical carcinoma-derived HeLa cells have been successfully used in plasmid expression systems to study the interaction between SV41 HN and F proteins . Other cell lines including mouse L929 cells and baby hamster kidney-derived BHK cells maintained in Eagle's minimum essential medium (MEM) supplemented with 5% calf serum have also demonstrated utility in these studies .

For expression systems, the plasmid vector pcDL-SRα296 (SRα) has been effectively employed, where gene expression is controlled by the SV40 early promoter and/or R-U5 sequence of the human T-cell leukemia virus type 1 long terminal repeat . This system facilitates robust expression of recombinant SV41 HN protein, enabling detailed functional analyses. When designing expression constructs, researchers should incorporate appropriate epitope tags or fluorescent markers to facilitate detection and purification without compromising protein function.

How can researchers generate chimeric constructs of SV41 HN for structure-function studies?

To generate chimeric constructs of SV41 HN for structure-function analyses, researchers should follow this methodological approach:

• Design and primer preparation: Identify regions of interest for chimera construction based on sequence alignments between SV41 HN and other paramyxovirus HN proteins. Design primers incorporating appropriate restriction sites to facilitate subsequent cloning steps.

• Site-directed mutagenesis: Introduce restriction sites (such as HindIII, BbeI, SacII, SplI, and VspI) into recombinant plasmids encoding SV41 HN using site-directed mutagenesis . This creates strategic cutting points for domain swapping.

• Domain exchange: Digest plasmids with appropriate restriction enzymes and ligate fragments to create chimeric constructs.

• Verification: Confirm chimeric structures through direct nucleotide sequencing using genetic analyzers such as the ABI PRISM 310 .

• Expression validation: Transfect the chimeric constructs into appropriate cell lines and verify expression through Western blotting or immunofluorescence using specific antibodies.

This approach has been successfully applied to create chimeras between various viral proteins, including F proteins of PIV5 and SV41, providing insights into domain-specific functions .

What methodologies are most effective for studying SV41 HN-F protein interactions?

Several complementary methodologies have proven effective for investigating SV41 HN-F protein interactions:

• Cell-cell fusion assays: Co-express SV41 HN with various F proteins in cell culture systems and quantify fusion activity through syncytia formation or reporter gene expression. This approach has revealed that SV41 F protein induces cell fusion when coexpressed with PIV-2 HN protein, while PIV-2 F protein does not induce fusion with SV41 HN .

• Co-immunoprecipitation: Use specific antibodies against SV41 HN or F proteins to precipitate protein complexes and analyze interacting partners by Western blotting. Anti-peptide rabbit sera specific for viral protein sequences can be prepared for this purpose .

• Chimeric protein analysis: Generate chimeric F proteins containing domains from different viruses to map regions required for HN interaction. Studies have shown that replacement of specific domains in the PIV5 F protein with SV41 F counterparts confers the ability to induce cell-cell fusion when coexpressed with SV41 HN .

• Site-directed mutagenesis: Introduce point mutations in potential interaction regions to identify specific amino acids critical for HN-F interaction. Research has demonstrated that replacement of as few as 21 amino acids in the PIV5 F protein with SV41 F-protein counterparts is sufficient to convert its HN protein specificity .

• Surface plasmon resonance: Measure binding kinetics and affinity between purified HN and F proteins to quantify interaction strength under various conditions.

The combination of these approaches provides comprehensive insights into the molecular determinants of HN-F interactions and their role in mediating membrane fusion.

How does the HN-F protein interaction trigger the membrane fusion process in paramyxoviruses?

The HN-F protein interaction in paramyxoviruses, including SV41, orchestrates a complex cascade of conformational changes that culminate in membrane fusion. The current model, supported by experimental evidence, suggests the following sequence:

• Receptor binding: HN protein binds to sialic acid-containing receptors on the target cell membrane.

• Conformational change in HN: Receptor binding induces a conformational change in HN, which triggers its interaction with the F protein .

• F protein activation: The HN-F interaction destabilizes the metastable prefusion conformation of F protein, leading to the insertion of the fusion peptide into the target membrane.

• Formation of the HR1 coiled-coil: The heptad repeat 1 (HR1) domain of the F protein forms a trimeric coiled-coil structure, extending the fusion peptide toward the target membrane .

• HR2 refolding: The heptad repeat 2 (HR2) domain folds back to form a six-helix bundle with HR1, bringing the viral and cellular membranes into close proximity.

• Membrane fusion: The physical force generated by the six-helix bundle formation drives membrane merger, initially creating a hemifusion intermediate followed by full fusion pore formation.

This process requires precise timing and coordination between HN and F proteins. For most paramyxoviruses (except SV5 strain W3A), HN undergoes a conformational change upon receptor binding, which then triggers the F protein conformational change through their specific interaction . The intervening sequence between HR1 and HR2 domains in the F protein is unusually long, a characteristic feature of paramyxovirus F proteins that facilitates the complex conformational changes required during the fusion process .

Which specific domains of SV41 HN determine its F protein specificity?

The specificity of interaction between SV41 HN and its cognate F protein is determined by multiple structural domains within the HN protein. While the search results don't provide explicit mapping of SV41 HN domains, the complementary studies on F protein specificity offer valuable insights.

Research on chimeric F proteins has revealed that specific domains in the F protein determine its ability to interact with SV41 HN. For example, studies have identified two regions (designated M1 and M2) on the PIV-2 F protein, either of which was necessary for chimeric F proteins to show fusogenic activity with the MuV HN protein. Additionally, two other regions (P1 and P2) were identified on the PIV-2 F protein, both of which were necessary to prevent induction of cell fusion with the SV41 HN protein .

These findings suggest that the corresponding interaction domains on SV41 HN likely contain structural features that specifically recognize these regions on the F protein. Future studies using similar chimeric approaches with the HN protein could precisely map these interaction domains.

How can chimeric SV41 HN-F proteins be used to study virus-specific fusion mechanisms?

Chimeric SV41 HN-F proteins represent powerful tools for dissecting virus-specific fusion mechanisms through several systematic approaches:

• Domain mapping: By creating chimeras where specific domains of SV41 HN or F proteins are replaced with corresponding regions from other paramyxoviruses (e.g., PIV5, PIV-2, MuV), researchers can identify critical regions that determine fusion specificity. Previous studies have shown that replacing five domains in the head region of the PIV5 F protein with SV41 F counterparts converted it to an SV41 HN-specific chimeric F protein .

• Fine mapping through segmentation: After identifying critical domains, these regions can be further divided into smaller segments to pinpoint specific interaction sites. For example, dividing five SV41 F-protein-derived domains into 16 segments revealed that 9 of these segments were not involved in determining specificity for the SV41 HN protein .

• Mutational analysis: Perform site-directed mutagenesis on chimeric proteins to identify individual amino acids crucial for specificity. Research has demonstrated that replacement of at most 21 amino acids of the PIV5 F protein with SV41 F-protein counterparts was sufficient to convert its HN protein specificity .

• Fusion assays with chimeric proteins: Express various chimeric constructs in cell culture systems and quantify their fusion activity through syncytia formation or reporter gene activation to correlate structural features with functional outcomes.

This systematic approach has yielded significant insights, including the observation that region M2 on the PIV-2 F protein is located at the membrane proximal end of the F1 ectodomain, while regions P1, M1, and P2 cluster together in the middle of the ectodomain . These regions may be involved in the functional interaction with HN proteins that is prerequisite for cell fusion.

How might recombinant SV41 HN be used in the development of viral vectors for gene therapy?

Recombinant SV41 HN protein has significant potential for viral vector development in gene therapy applications through several strategic approaches:

• Targeted entry vectors: By engineering viral vectors to express SV41 HN with modified receptor specificity, researchers could develop systems that selectively target specific cell types. This approach could enhance the precision of gene delivery to tissues of interest while minimizing off-target effects.

• Fusogenic enhancement: Incorporating SV41 HN along with compatible F proteins into viral vectors could enhance membrane fusion capabilities, potentially improving the efficiency of gene delivery into target cells. The virus type-specific interaction between HN and F proteins provides a tunable system for controlling fusion activity .

• Immune evasion strategies: Understanding the immunogenic properties of SV41 HN could inform the development of vectors with reduced immunogenicity. By identifying and modifying highly immunogenic epitopes while preserving functional domains, researchers could create vectors capable of repeated administration without neutralization.

• Chimeric vector development: Knowledge gained from chimeric analyses of SV41 HN and related proteins could guide the creation of hybrid vectors with optimized properties. For instance, chimeric F proteins containing domains from different viruses have demonstrated altered fusion specificities , suggesting that similar approaches with HN could yield vectors with tailored cell entry mechanisms.

The development of such vectors would benefit from the methodological approaches used in basic SV41 HN research, including chimeric protein design, site-directed mutagenesis, and cell-cell fusion assays to validate vector function.

What are the current challenges in expressing and purifying functional recombinant SV41 HN protein for structural studies?

Expressing and purifying functional recombinant SV41 HN protein for structural studies presents several significant challenges:

• Maintaining native conformation: As a complex glycoprotein with multiple functional domains, SV41 HN requires proper folding and post-translational modifications to maintain its native conformation. Expression systems must preserve these structural features to ensure functional relevance.

• Glycosylation requirements: The HN protein contains multiple N-linked glycosylation sites that influence its folding, stability, and activity. Different expression systems (mammalian, insect, yeast) produce varying glycosylation patterns, potentially affecting protein function and structure.

• Membrane association: SV41 HN is a type II membrane protein with a transmembrane domain that anchors it to the viral envelope. For structural studies, researchers must decide whether to express the full-length protein (requiring detergent solubilization) or a truncated, soluble form (potentially missing key structural elements).

• Protein stability: The HN protein may exhibit conformational instability during purification procedures, particularly when removed from its membrane environment. Stabilizing agents or engineering strategies may be required to maintain the protein in a suitable state for crystallization or cryo-EM studies.

• Functional validation: Ensuring that the purified protein retains both hemagglutinin and neuraminidase activities is essential for meaningful structural analysis. Functional assays must be incorporated into the purification workflow to validate the integrity of the protein.

Addressing these challenges requires a multifaceted approach, potentially combining mammalian expression systems (like HeLa or BHK cells ) for proper folding and post-translational modifications, optimized solubilization strategies for membrane extraction, and rigorous functional validation to ensure the purified protein accurately represents its native state.

How have genetic analyses of SV41 HN contributed to understanding paramyxovirus evolution?

Genetic analyses of SV41 HN have provided crucial insights into paramyxovirus evolution through several key contributions:

• Sequence conservation patterns: Comparative sequence analyses between SV41 HN and other paramyxovirus HN proteins have revealed highly conserved regions that likely perform essential functions maintained throughout evolution. These regions include the neuraminidase active site and domains involved in protein folding and stability.

• Functional domain evolution: Studies of chimeric F proteins interacting with SV41 HN have demonstrated that specific domains determine virus type-specific interactions . These findings suggest that co-evolution of HN and F proteins has occurred within viral species, with selective pressures maintaining compatible interaction interfaces while allowing divergence in other regions.

• Host adaptation signatures: Analysis of SV41 HN sequences may reveal signatures of adaptation to specific host cell receptors. These adaptive changes would reflect the evolutionary history of host-virus interactions and potentially explain host range limitations.

• Recombination events: The observation that replacement of specific domains in the F protein can change its specificity for HN proteins suggests that recombination events between related viruses could have played a role in paramyxovirus evolution. Similar recombination events involving HN genes could contribute to viral diversification and adaptation.

These genetic analyses contribute to our understanding of the evolutionary relationships among paramyxoviruses and provide insights into the molecular mechanisms driving viral adaptation and speciation.

What insights have been gained from comparing the fusion mechanisms of SV41 with those of other paramyxoviruses?

Comparative studies of fusion mechanisms between SV41 and other paramyxoviruses have yielded several significant insights:

• HN-dependent vs. HN-independent fusion: While most paramyxoviruses (including SV41) require a virus type-specific interaction between HN and F proteins to mediate fusion, some viruses like SV5 strain W3A can induce cell fusion through their F protein alone, independent of HN protein . This distinction highlights evolutionary divergence in fusion strategies.

• Virus-specific interactions: Research has revealed strict specificity in HN-F interactions that determine fusion capabilities. For example:

  • SV41 F protein induces cell fusion with PIV-2 HN protein

  • PIV-2 F protein cannot induce fusion with SV41 HN protein

  • PIV-2 F protein induces fusion with MuV HN protein

  • SV41 F protein does not induce fusion with MuV HN protein

  These patterns demonstrate the complex co-evolution of viral fusion machinery.

• Domain-specific determinants: Chimeric studies have identified specific regions in F proteins that determine HN-specificity. Regions P1 and P2 on the PIV-2 F protein prevent fusion with SV41 HN, while regions M1 and M2 enable fusion with MuV HN . These regions cluster in specific locations on the F protein, suggesting structural conservation of interaction interfaces across different paramyxoviruses.

• Conformational change mechanisms: For most paramyxoviruses, the current model suggests that HN undergoes a conformational change upon receptor binding, which triggers F protein activation through specific interactions . This sequential activation system appears to be conserved across most paramyxoviruses, with variations in the specific molecular details that determine virus-type specificity.

These comparative insights enhance our understanding of the evolutionary relationships among paramyxoviruses and provide a framework for predicting and potentially manipulating fusion behaviors in recombinant viral systems.

How might recombinant SV41 HN contribute to novel vaccine strategies against paramyxovirus infections?

Recombinant SV41 HN protein offers several promising avenues for novel vaccine development against paramyxovirus infections:

• Chimeric antigen design: Knowledge gained from chimeric studies of SV41 HN with other paramyxovirus proteins can inform the design of immunogens that present conserved neutralizing epitopes while eliminating regions that induce non-neutralizing or potentially harmful immune responses. These rationally designed antigens could elicit broader protection against multiple paramyxovirus species.

• Vector-based vaccines: Recombinant viral vectors expressing SV41 HN could serve as effective vaccine platforms. The specificity of HN-F interactions could be exploited to develop vectors with controlled fusogenic properties, potentially enhancing immunogenicity through improved antigen presentation or cellular delivery.

• Structure-based immunogen design: Detailed structural understanding of SV41 HN, particularly its receptor-binding sites and epitopes targeted by neutralizing antibodies, could guide the design of stabilized immunogens that present these critical regions in their native conformations.

• Adjuvant properties: The neuraminidase activity of SV41 HN could potentially serve as a natural adjuvant by modifying cell surface glycans and thereby modulating immune cell activation. This property could be harnessed in vaccine formulations to enhance immunogenicity.

• Cross-protective immunity: By identifying conserved epitopes between SV41 HN and related human paramyxovirus HN proteins, researchers could develop immunization strategies targeting these regions to induce cross-protective immunity against multiple viral pathogens.

These approaches draw upon the fundamental research on SV41 HN structure, function, and interactions to develop next-generation vaccines with improved efficacy and potentially broader protection against paramyxovirus infections.

What are the experimental considerations for testing recombinant SV41 HN-based vaccines in animal models?

Testing recombinant SV41 HN-based vaccines in animal models requires careful consideration of multiple experimental parameters:

• Model selection: Choose appropriate animal models that support SV41 infection or that have receptors compatible with SV41 HN binding. Non-human primates represent ideal models due to their phylogenetic proximity to humans, but smaller animal models may be necessary for initial studies .

• Immunization protocol design: Consider heterologous prime-boost strategies that combine different delivery platforms (e.g., recombinant protein, viral vectors, DNA) to enhance immune responses. Previous studies with recombinant simian virus vaccines have successfully utilized this approach, with immunizations administered at multiple time points (e.g., 0, 8, 24, 43, and 54 weeks) .

• Antigen formulation: Determine optimal adjuvant selection, dosage, and route of administration for recombinant SV41 HN proteins. The addition of complementary viral antigens, such as the F protein, may enhance protection by targeting multiple components of the viral entry machinery.

• Immune response assessment: Employ comprehensive immunological assays to evaluate:

  • Antibody responses (neutralizing and binding antibodies)

  • T cell responses (CD4+ and CD8+ T cells)

  • Mucosal immunity (particularly for respiratory paramyxoviruses)

  • Duration of immunity through longitudinal sampling

• Challenge studies: Design appropriate viral challenge protocols that mimic natural infection routes. For respiratory paramyxoviruses, this typically involves intranasal or aerosol challenge.

• Safety evaluation: Monitor for potential adverse effects, including enhanced respiratory disease that has been associated with some paramyxovirus vaccine candidates.

• Correlates of protection: Identify immunological markers that correlate with protection, which could inform future vaccine development and regulatory approval pathways.

Successful implementation of these considerations would generate valuable data on the potential efficacy and safety of recombinant SV41 HN-based vaccines, potentially paving the way for similar approaches with human paramyxovirus antigens.

What novel technologies could advance our understanding of SV41 HN structure-function relationships?

Several emerging technologies hold promise for deepening our understanding of SV41 HN structure-function relationships:

• Cryo-electron microscopy (cryo-EM): Recent advances in cryo-EM technology now enable near-atomic resolution structures of membrane proteins like SV41 HN, potentially revealing conformational changes upon receptor binding and interactions with F protein.

• Single-molecule fluorescence resonance energy transfer (smFRET): This technique could track real-time conformational changes in SV41 HN during receptor binding and F protein interaction, providing dynamic information not accessible through static structural methods.

• AlphaFold and other AI-based structure prediction: These computational approaches could predict SV41 HN structures in different conformational states and in complex with F protein, generating testable hypotheses about interaction interfaces.

• CRISPR-based mutagenesis screens: Systematic mutagenesis of SV41 HN combined with functional assays could comprehensively map functional domains and critical residues across the entire protein.

• Native mass spectrometry: This technique could analyze the composition and stoichiometry of HN-F protein complexes in their native state, providing insights into the assembly of functional fusion complexes.

• Super-resolution microscopy: Advanced imaging techniques could visualize the spatial and temporal dynamics of HN-F interactions during the fusion process in living cells, revealing the coordinated sequence of events leading to membrane fusion.

• Glycomics approaches: Comprehensive analysis of SV41 HN glycosylation patterns and their impact on protein function could reveal how post-translational modifications modulate receptor binding and fusion promotion activities.

Implementation of these technologies could resolve long-standing questions about how SV41 HN orchestrates the complex process of membrane fusion through its interactions with cellular receptors and the viral F protein.

How might the study of SV41 HN contribute to understanding broader principles of viral membrane fusion?

The study of SV41 HN offers unique opportunities to advance our understanding of broad principles governing viral membrane fusion:

• Regulated fusion activation: SV41 represents a model system for studying how protein-protein interactions (HN-F) regulate the timing and location of fusion activation. Unlike some viruses where fusion is triggered solely by receptor binding or pH changes, paramyxoviruses employ a two-protein system with additional regulatory complexity . This system provides insights into how viruses ensure fusion occurs at the right time and place.

• Co-evolution of fusion machinery: The virus type-specific interaction between HN and F proteins highlights principles of co-evolution within viral fusion machinery. Studying these specific interactions can reveal how paired fusion systems evolve while maintaining functional compatibility .

• Conformational coupling mechanisms: Understanding how conformational changes propagate from HN to F protein can illuminate general principles of allosteric regulation in protein complexes. This knowledge extends beyond virology to basic protein science.

• Receptor-mediated activation pathways: For most paramyxoviruses, HN undergoes conformational changes upon receptor binding, which then trigger F protein activation . This cascade exemplifies how receptor engagement can be translated into mechanical force for membrane fusion through a series of protein conformational changes.

• Fusion specificity determinants: Chimeric studies revealing that replacement of specific domains can alter fusion specificity demonstrate how modular protein domains encode interaction specificity. These principles apply broadly to protein-protein interactions beyond viral fusion.

• Metastability in fusion proteins: The F protein exists in a metastable prefusion conformation that is triggered to undergo dramatic conformational changes during fusion . SV41 studies contribute to understanding how this metastability is maintained and regulated, a feature common to many viral fusion systems.