Encephalitis Japanese 12kda

What is the Japanese encephalitis virus 12kDa protein?

The Japanese encephalitis virus 12kDa protein refers to the viral capsid (C) protein, one of the three structural proteins encoded by the JEV genome. JEV belongs to the flavivirus family, closely related to West Nile, yellow fever, and dengue viruses. The JEV genome is a positive-stranded RNA that is translated as a single polyprotein and subsequently cleaved by host and viral proteases into structural proteins (C, prM/M, and E) and at least seven nonstructural proteins (NS1/NS1', NS2A, NS2B, NS3, NS4A, NS4B, and NS5) . The capsid protein specifically binds to viral RNA to form a nucleocapsid that is then enveloped by an endoplasmic reticulum-derived membrane containing the envelope (E) and premembrane (prM) proteins .

The recombinant form of this protein typically contains 110 amino acids with a molecular weight of 12kDa. For research applications, it is often produced with fusion tags (such as His-tag) to facilitate purification and downstream applications . The native protein plays essential roles in virus assembly and potentially in interactions with host cellular factors during infection.

What experimental methods are used to express and purify the JEV 12kDa protein?

The expression and purification of JEV 12kDa protein can be accomplished through several methodological approaches:

• Bacterial Expression System: The protein can be recombinantly produced in E. coli expression systems, typically fused to a His-tag at the N-terminus to facilitate purification . This approach is cost-effective and yields high protein quantities, although it lacks eukaryotic post-translational modifications.

• Mammalian Expression System: For studies requiring native folding and post-translational modifications, the protein can be expressed in mammalian cells such as HEK293 cells, similar to approaches used for other JEV proteins . This method preserves the protein's native folding state and maintains all post-translational modifications.

• Purification Protocol:

  • Affinity chromatography using nickel columns for His-tagged proteins

  • Proprietary chromatographic techniques for higher purity (>95% as determined by PAGE with Coomassie staining)

  • Buffer formulation typically includes PBS with stabilizing agents such as K₂CO₃

• Quality Control:

  • SDS-PAGE analysis for purity assessment

  • Western blotting for identity confirmation

  • Functional assays to confirm biological activity

For long-term storage of the purified protein, addition of carrier proteins (0.1% HSA or BSA) is recommended to maintain stability, and multiple freeze-thaw cycles should be avoided .

How does the JEV 12kDa protein function in the viral lifecycle?

The 12kDa capsid protein performs several critical functions in the JEV lifecycle:

• Nucleocapsid Formation: The primary function of the capsid protein is to bind viral genomic RNA, forming the nucleocapsid that protects the viral genetic material . This RNA-protein complex is essential for virion assembly and stability.

• Structural Role: The protein provides structural support for the viral particle by creating an organized scaffold around which the envelope and membrane proteins are arranged.

• Viral Assembly: During viral replication, the capsid protein coordinates the assembly process by interacting with both viral RNA and the structural proteins embedded in the endoplasmic reticulum membrane.

• Potential Host Interactions: Though not explicitly detailed in the search results, research on related flaviviruses suggests that capsid proteins may interact with host factors to modulate cellular processes and potentially contribute to immune evasion mechanisms.

Experimental approaches to study these functions include:

• Mutagenesis studies to identify functional domains

• RNA binding assays to characterize nucleic acid interactions

• Protein-protein interaction studies using co-immunoprecipitation

• Subcellular localization analysis using immunofluorescence microscopy

Understanding these functions provides insights into potential targets for antiviral development and basic mechanisms of flavivirus biology.

What methods are used to detect the JEV 12kDa protein in experimental settings?

Several methodological approaches are employed to detect and quantify the JEV 12kDa capsid protein:

• Immunoassays:

  • ELISA (Enzyme-Linked Immunosorbent Assay): Recombinant 12kDa protein can be used as a standard or coating antigen

  • Western Blot: Using specific antibodies against the capsid protein, often with enhanced chemiluminescence detection

  • Immunoprecipitation: For studying protein-protein interactions

• Molecular Detection Methods:

  • SDS-PAGE with Coomassie staining for purified protein

  • Mass spectrometry for protein identification and characterization

  • Protein electrophoresis coupled with immunoblotting

• Immunohistochemistry/Immunofluorescence:

  • For detecting the protein in infected tissues or cells

  • Often combined with confocal microscopy for subcellular localization studies

• Flow Cytometry:

  • For detecting intracellular protein in single cells

  • Useful for studying infection rates and protein expression levels

The choice of detection method depends on the research question, sample type, and required sensitivity. For instance, in studies examining DAP12 phosphorylation in JEV-infected macrophages, immunoprecipitation followed by immunoblotting with anti-phosphotyrosine antibodies has been employed to investigate signaling pathways .

What are the stability considerations when working with the JEV 12kDa protein?

Maintaining the stability of the JEV 12kDa protein is critical for experimental reproducibility and validity. Key considerations include:

• Storage Conditions:

  • Short-term storage (2-4 weeks): 4°C if the entire preparation will be used

  • Long-term storage: -20°C with appropriate stabilizing additives

  • Addition of carrier proteins (0.1% HSA or BSA) is recommended for long-term storage

• Buffer Composition:

  • Commonly used formulations include PBS with 25mM K₂CO₃

  • pH stability is important, typically maintained at physiological range

  • Presence of reducing agents may be necessary depending on the structural characteristics

• Freeze-Thaw Stability:

  • Multiple freeze-thaw cycles should be avoided as they can lead to protein denaturation and aggregation

  • Aliquoting samples before freezing is recommended

• Physical Appearance:

  • Properly stored and formulated protein should maintain a sterile, filtered clear solution appearance

  • Precipitation or cloudiness may indicate denaturation or contamination

• Functional Stability:

  • Regular validation of protein activity using functional assays is recommended

  • Stability studies may be necessary for determining shelf-life in various applications

Following these guidelines ensures that the protein maintains its structural integrity and functional properties for reliable experimental outcomes in research settings.

How does the JEV 12kDa protein contribute to viral pathogenesis and neuroinflammation?

The contribution of the JEV 12kDa capsid protein to viral pathogenesis involves complex interactions with host factors and potential roles in neuroinflammation:

• Neuroinflammatory Mechanisms:
  While the specific role of the 12kDa protein in neuroinflammation is not explicitly detailed in the search results, JEV infection activates macrophages and microglia to secrete proinflammatory cytokines and chemokines, including TNF-α, IL-6, IL-18, and MCP-1 . These inflammatory mediators contribute to neuronal damage and blood-brain barrier (BBB) disruption.

• Experimental Approaches to Study Pathogenic Mechanisms:

  • In vitro neuronal cell models: To assess direct effects on neurons

  • Microglia activation assays: To measure inflammatory responses

  • BBB integrity assays: To evaluate disruption of the blood-brain barrier

  • Transcriptomic and proteomic analyses: To identify host factors affected by viral proteins

• Receptor Interactions:
  Research has identified that JEV interacts with C-type lectin receptor CLEC5A on macrophages and microglia, leading to DAP12 phosphorylation and subsequent inflammatory responses . Though this interaction has been established for the virus as a whole, specific investigations into the role of the capsid protein in these interactions would provide valuable insights.

• Experimental Data on Inflammatory Pathways:
  Studies have demonstrated that JEV infection induces DAP12 phosphorylation in macrophages, which can be detected through immunoprecipitation with anti-DAP12 antibodies followed by immunoblotting with anti-phosphotyrosine antibodies . This methodological approach could be applied to investigate potential capsid protein contributions to signaling pathway activation.

Understanding these mechanisms is crucial for developing targeted therapies that could mitigate JEV-induced neuroinflammation and associated neurological damage.

What structural analyses have been performed on the JEV 12kDa protein, and what methodologies are recommended?

Structural characterization of the JEV 12kDa capsid protein employs multiple complementary techniques:

• X-ray Crystallography:

  • Challenges include obtaining sufficient quantities of highly purified, homogeneous protein

  • Optimization of crystallization conditions may require screening hundreds of buffer compositions

  • Co-crystallization with RNA or host interaction partners can provide functional insights

• NMR Spectroscopy:

  • Useful for studying dynamic properties and binding interactions

  • Requires isotope-labeled protein (typically ¹⁵N, ¹³C)

  • Can provide atomic-level information about protein flexibility and conformational changes

• Cryo-Electron Microscopy:

  • Particularly valuable for visualizing the capsid in the context of whole virions

  • Can reveal structural arrangements without crystallization

  • Recent advances in resolution make this increasingly powerful for detailed structural analysis

• Computational Modeling:

  • Homology modeling based on related flavivirus capsid proteins

  • Molecular dynamics simulations to understand conformational changes

  • Docking studies to predict interactions with host factors or small molecules

• Protein Engineering Approaches:

  • Expression of recombinant protein with systematic mutations to identify functional domains

  • Production of truncated versions to study domain-specific functions

The current methodological recommendation is to combine multiple approaches to overcome limitations of individual techniques. For instance, recombinant JEV capsid protein can be produced with a C-terminal His-tag in HEK293 cells to maintain proper folding and post-translational modifications for structural studies , though E. coli expression systems may yield higher quantities for initial screening .

How can researchers investigate the interaction between JEV 12kDa protein and host immune system components?

Investigating interactions between the JEV 12kDa capsid protein and host immune components requires sophisticated methodological approaches:

• Receptor Binding Studies:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics with immune receptors

  • ELISA-based binding assays using purified recombinant 12kDa protein

  • Flow cytometry to detect binding to cell surface receptors on immune cells

• Signaling Pathway Activation:

  • Phosphorylation assays to detect activation of immune signaling pathways

  • Measurement of DAP12 phosphorylation, which has been identified in JEV infection

  • Reporter cell lines expressing specific immune receptors and signaling pathway reporters

• Cellular Response Analysis:

  • Cytokine profiling of macrophages and microglia exposed to purified 12kDa protein

  • Transcriptomic analysis to identify gene expression changes

  • Proteomic approaches to detect altered protein expression patterns

• In vitro Immune Cell Models:

  • Primary macrophage and microglial cultures

  • Differentiated cell lines (THP-1, BV-2)

  • Co-culture systems with neurons to assess bystander effects

• Methodological Protocol for Investigating Signaling Events:
  When studying phosphorylation events, researchers can follow this protocol:

  • Stimulate macrophages (1×10⁶) with JEV or purified 12kDa protein

  • Lyse cells in appropriate buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitor cocktail)

  • Perform immunoprecipitation with relevant antibodies (e.g., anti-DAP12)

  • Analyze by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies

  • Strip and reprobe membranes to detect total protein levels

These approaches allow for detailed characterization of how the 12kDa protein may contribute to immune activation or evasion during JEV infection.

What are the methodological considerations when designing experiments to evaluate the JEV 12kDa protein as a vaccine antigen?

Evaluating the JEV 12kDa capsid protein as a vaccine antigen requires careful experimental design with several methodological considerations:

• Antigen Preparation Strategies:

  • Recombinant protein expression systems: E. coli systems provide high yields but lack post-translational modifications

  • Mammalian expression systems (e.g., HEK293 cells): Provide protein in native folding state with appropriate post-translational modifications

  • Protein purification to >95% purity using chromatographic techniques

  • Verification of structural integrity through biophysical methods

• Immunogenicity Assessment Protocol:

  • Animal models: Selection of appropriate models that recapitulate human immune responses

  • Immunization schedules: Prime-boost regimens with optimized timing

  • Adjuvant selection: Critical for enhancing immune responses

  • Measurement of:

    • Antibody titers (total IgG and neutralizing antibodies)

    • T cell responses (proliferation, cytokine production, cytotoxicity)

    • Memory B and T cell development

• Protection Studies Design:

  • Challenge models: Selection of appropriate viral strains and challenge routes

  • Endpoints: Survival, viral load, clinical scores, histopathological assessment

  • Passive transfer experiments: To determine correlates of protection

• Immunological Analysis Methods:

  • ELISA for antibody responses

  • Neutralization assays for functional antibody assessment

  • ELISpot and intracellular cytokine staining for T cell responses

  • Flow cytometry for immune cell phenotyping

• Comparative Experimental Design:

  • Control groups: Adjuvant-only, unrelated antigen, established JEV vaccine

  • Dose-response studies: Multiple antigen concentrations

  • Antigen formulation variations: Soluble protein, particulate formulations, DNA vaccines

The recombinant JEV 12kDa protein has shown potential to elicit immune responses, making it a valuable tool for vaccine development. Research indicates that recombinant JEV proteins can induce neutralizing antibodies and cellular immune responses in animal models . A comprehensive evaluation would require assessment of both humoral and cellular immunity to determine protective efficacy against viral challenge.

How can researchers design studies to investigate the role of the JEV 12kDa protein in blood-brain barrier disruption?

Investigating the role of the JEV 12kDa capsid protein in blood-brain barrier (BBB) disruption requires sophisticated experimental designs and methodologies:

• In vitro BBB Models:

  • Human brain microvascular endothelial cell (HBMEC) monolayers

  • Transwell co-culture systems with astrocytes and pericytes for more physiologically relevant models

  • Measurement parameters:

    • Transendothelial electrical resistance (TEER)

    • Permeability to fluorescent tracers (FITC-dextran, sodium fluorescein)

    • Expression of tight junction proteins (ZO-1, claudin-5, occludin)

• Mechanistic Studies Protocol:

  • Treatment of BBB models with purified 12kDa protein at various concentrations

  • Assessment of direct effects versus indirect effects mediated by inflammatory responses

  • Analysis of signaling pathways involved (PKC, Rho GTPases, MAPK)

  • Evaluation of:

    • Cytokine/chemokine production by ELISA or multiplex assays

    • Tight junction protein localization by immunofluorescence

    • Actin cytoskeleton rearrangement

    • Matrix metalloproteinase (MMP) activation

• In vivo BBB Integrity Assessment:

  • Animal models with:

    • Evans blue extravasation assay

    • Magnetic resonance imaging with contrast agents

    • Immunohistochemical analysis of tight junction proteins

  • Experimental approaches:

    • Direct injection of purified 12kDa protein

    • Use of genetically modified viruses expressing mutated capsid protein

    • Temporal correlation between capsid protein levels and BBB disruption

• Comparison with Known BBB Disruptors:

  • Control experiments with TNF-α or IL-1β as positive controls

  • Comparison with other viral proteins known to disrupt BBB

  • Neutralization experiments with antibodies against the 12kDa protein

Research has demonstrated that JEV infection causes BBB disintegrity in STAT1-deficient mice, and peripheral administration of antibodies targeting host receptors (e.g., anti-CLEC5A mAb) can restore BBB integrity and reduce infiltration of virus-harboring leukocytes into the CNS . Similar experimental approaches could be applied to investigate the specific contribution of the 12kDa capsid protein to this pathological process.

What methodological approaches can researchers use to identify small molecule inhibitors targeting the JEV 12kDa protein?

Identifying small molecule inhibitors of the JEV 12kDa capsid protein involves a multi-faceted drug discovery approach:

• High-Throughput Screening (HTS) Methods:

  • Biochemical assays:

    • Fluorescence-based thermal shift assays to detect protein stabilization

    • AlphaScreen technology for detecting protein-protein or protein-RNA interactions

  • Cell-based assays:

    • Reporter virus systems

    • Viral replication inhibition assays

    • Cytopathic effect (CPE) reduction assays

• Structure-Based Drug Design Protocol:

  • In silico docking studies using 3D structures of the capsid protein

  • Molecular dynamics simulations to identify binding pockets

  • Fragment-based screening

  • Virtual compound library screening

• Target Validation Approaches:

  • Mutagenesis of potential binding sites

  • Competitive binding assays with known ligands

  • NMR or X-ray crystallography to confirm binding mode

• Lead Optimization Strategy:

  • Structure-activity relationship (SAR) studies

  • Medicinal chemistry modifications to improve:

    • Binding affinity

    • Selectivity

    • Cell permeability

    • Metabolic stability

    • Blood-brain barrier penetration

• Functional Validation Methods:

  • Validation of mechanism of action:

    • Viral assembly assays

    • Nucleocapsid formation assays

    • RNA binding inhibition assays

  • Specificity testing against other flavivirus capsid proteins

  • Testing in various cell types including neurons and glial cells

• In vivo Efficacy Assessment:

  • Pharmacokinetic profiling with focus on CNS penetration

  • Efficacy testing in animal models of JEV infection

  • Assessment of reduction in viral load and neuroinflammation

Given that the JEV capsid protein plays critical roles in viral assembly and potentially in pathogenesis, targeting it with small molecules represents a promising antiviral strategy. The availability of purified recombinant protein facilitates the implementation of these drug discovery approaches.

How can the JEV 12kDa protein be utilized in diagnostic assay development?

The JEV 12kDa capsid protein offers several applications in diagnostic assay development:

• Serological Assay Development:

  • Recombinant 12kDa protein can serve as a capture antigen in ELISA-based systems

  • Methodological considerations for assay optimization:

    • Protein coating concentration optimization (typically 1-10 μg/ml)

    • Blocking buffer selection to minimize background

    • Sample dilution protocols for optimal sensitivity and specificity

    • Secondary antibody selection and optimization

• Multiplex Assay Integration:

  • Inclusion in multiplex platforms for differential diagnosis of flavivirus infections

  • Bead-based assays (e.g., Luminex technology)

  • Protein microarray applications

• Point-of-Care Diagnostic Development Protocol:

  • Lateral flow immunoassay design using 12kDa protein

  • Gold nanoparticle conjugation methods

  • Stability testing under various environmental conditions

  • Sensitivity and specificity optimization

• Molecular Detection Applications:

  • Development of standards for nucleic acid amplification tests

  • Positive controls for PCR or isothermal amplification methods

  • Internal controls for diagnostic assays

• Performance Evaluation Methodology:

  • Cross-reactivity assessment with other flaviviruses

  • Sensitivity and specificity determination

  • Reproducibility and robustness testing

  • Field evaluation in endemic regions

The recombinant JEV 12kDa protein, with its high purity (>95% as determined by PAGE with Coomassie staining) , provides a standardized reagent for diagnostic development. Its suitability for ELISA applications has been demonstrated , making it a valuable tool for both research and clinical diagnostic applications.

What research gaps exist in understanding the interaction between the JEV 12kDa protein and host cellular factors?

Several important research gaps remain in understanding the interactions between the JEV 12kDa capsid protein and host cellular factors:

• Protein-Protein Interaction Networks:

  • Comprehensive interactome mapping of capsid-host protein interactions

  • Methodological approaches needed:

    • Proximity labeling techniques (BioID, APEX)

    • Co-immunoprecipitation coupled with mass spectrometry

    • Yeast two-hybrid or mammalian two-hybrid screening

    • Protein complementation assays

• Subcellular Localization and Trafficking:

  • Dynamics of capsid protein localization during infection

  • Research techniques to address:

    • Live-cell imaging with fluorescently tagged proteins

    • Time-course immunofluorescence studies

    • Fractionation studies to identify compartment-specific interactions

• Post-Translational Modifications:

  • Characterization of modifications on the capsid protein and their functional significance

  • Methodological needs:

    • Mass spectrometry-based proteomic approaches

    • Site-directed mutagenesis of modification sites

    • Functional assays to assess the impact of modifications

• Role in Immune Evasion:

  • Mechanisms by which the capsid protein might antagonize host immune responses

  • Experimental approaches:

    • Innate immune signaling reporter assays

    • Analysis of interferon pathway components

    • Immunoprecipitation studies with key immune factors

• Contributions to Neurotropism:

  • How the capsid protein might influence JEV's neurotropic properties

  • Research strategies:

    • Neuron-specific interaction studies

    • Comparative analyses with non-neurotropic flaviviruses

    • Generation of chimeric viruses with capsid protein exchanges

While research has established that JEV interacts with host receptors like CLEC5A to trigger inflammatory responses , the specific contribution of the 12kDa capsid protein to these interactions and other host processes remains to be fully elucidated. Addressing these gaps will require integrated approaches combining structural biology, cellular studies, and in vivo models.

How can researchers design experiments to compare the 12kDa protein across different JEV strains and related flaviviruses?

Comparative analysis of the 12kDa capsid protein across JEV strains and related flaviviruses requires systematic experimental design:

• Sequence and Structural Comparative Analysis Protocol:

  • Multiple sequence alignment of capsid proteins from:

    • Different JEV genotypes (I-V)

    • Related flaviviruses (West Nile, dengue, yellow fever)

  • Identification of:

    • Conserved regions (potential functional domains)

    • Variable regions (potential strain-specific functions)

    • Phylogenetic relationship analysis

• Functional Conservation Assessment Methodology:

  • Recombinant protein production of multiple variants

  • Comparative functional assays:

    • RNA binding efficiency

    • Protein-protein interaction profiles

    • Nuclear localization efficiency

    • Effects on host cell processes

• Antigenic Comparison Design:

  • Generation of strain-specific and cross-reactive antibodies

  • Epitope mapping techniques:

    • Peptide arrays

    • Phage display

    • Hydrogen-deuterium exchange mass spectrometry

  • Cross-neutralization studies

• Chimeric Protein Approaches:

  • Design and production of chimeric capsid proteins

  • Domain swapping experiments to identify functional regions

  • Assessment in mini-genome systems or replicon models

• Cross-Species Pathogenesis Studies:

  • In vitro comparative studies using cells from different host species

  • Analysis of species-specific interactions with host factors

  • Correlation with host range and pathogenicity

The SA-14 strain of JEV has been used as a reference for recombinant protein production (NCBI Accession Number: P27395.1) , providing a starting point for comparative studies. Systematic comparison across strains would yield valuable insights into conserved functional mechanisms and strain-specific characteristics that might contribute to differences in virulence or host range.

What methodological considerations are important when studying the role of the JEV 12kDa protein in viral RNA binding?

Studying the RNA binding properties of the JEV 12kDa capsid protein requires specialized methodological approaches:

• RNA-Protein Interaction Assay Methods:

  • Electrophoretic Mobility Shift Assay (EMSA):

    • Using labeled viral RNA fragments

    • Titration with increasing protein concentrations

    • Competition assays with specific and non-specific RNA

  • Filter Binding Assays:

    • Quantitative measurement of RNA binding affinities

    • Determination of binding kinetics

  • Surface Plasmon Resonance (SPR):

    • Real-time binding kinetics measurement

    • Association and dissociation rate determination

• RNA Structure and Binding Site Mapping Protocol:

  • SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension)

  • Hydroxyl radical footprinting

  • RNA immunoprecipitation followed by sequencing (RIP-seq)

  • CLIP (Cross-linking and immunoprecipitation) methods

• Mutational Analysis Design:

  • Alanine scanning mutagenesis of basic residues

  • Structure-guided mutations of predicted RNA-binding domains

  • Charge reversal mutations

  • Assessment of mutants in binding assays and functional studies

• Biophysical Characterization Methods:

  • Isothermal Titration Calorimetry (ITC):

    • Thermodynamic parameters of binding

    • Stoichiometry determination

  • Circular Dichroism (CD):

    • Conformational changes upon RNA binding

    • Secondary structure analysis

  • Fluorescence spectroscopy:

    • Intrinsic tryptophan fluorescence changes

    • FRET-based assays for binding dynamics

• In-Cell RNA Binding Assessment:

  • RNA immunoprecipitation from infected cells

  • Fluorescence in situ hybridization (FISH)

  • Proximity ligation assays

  • Live-cell imaging with labeled components

The JEV capsid protein binds to viral RNA to form a nucleocapsid , a critical step in virion assembly. Understanding the molecular details of this interaction could provide insights into potential targets for antiviral development. The availability of recombinant capsid protein facilitates these mechanistic studies by providing a defined starting material for in vitro analyses.

What are the challenges in differentiating between the JEV 12kDa protein and similar proteins from related flaviviruses?

Distinguishing the JEV 12kDa capsid protein from homologous proteins of related flaviviruses presents several technical challenges with corresponding methodological solutions:

• Cross-Reactivity in Immunological Detection:

  • Challenge: Antibodies against the JEV capsid may cross-react with other flavivirus capsid proteins due to sequence similarity

  • Methodological solutions:

    • Epitope mapping to identify JEV-specific regions

    • Development of monoclonal antibodies targeting unique epitopes

    • Competitive ELISA designs with blocking steps

    • Differential absorption techniques to improve specificity

• Sequence and Structural Similarity:

  • Challenge: High conservation in certain domains complicates distinction based on sequence or structure alone

  • Technical approaches:

    • High-resolution mass spectrometry for peptide fingerprinting

    • Targeted proteomics focusing on unique peptide sequences

    • Structural analysis of subtle conformational differences

• Functional Assay Specificity:

  • Challenge: Similar functional properties across flavivirus capsid proteins

  • Methodological strategies:

    • Comparative functional assays under varying conditions

    • Use of host factor interactions unique to JEV capsid

    • Development of JEV-specific reporter systems

• Diagnostic Application Challenges:

  • Challenge: False positives in endemic areas with multiple circulating flaviviruses

  • Technical solutions:

    • Multiplex assays with algorithmic interpretation

    • Sequential testing strategies

    • Statistical approaches to interpret results in high cross-reactivity scenarios

• Experimental Design for Specificity Testing:

  • Panels of recombinant capsid proteins from multiple flaviviruses

  • Systematic cross-reactivity assessment

  • Quantitative analysis of binding affinities and kinetics

  • Creation of chimeric proteins to map specificity determinants

The recombinant JEV envelope protein has been manufactured to address needs for highly purified, concentrated protein for vaccine research and serological based diagnostic products . Similar approaches for the capsid protein would facilitate development of more specific detection methods and improve differentiation from related flavivirus proteins.

What are the methodological considerations when designing experiments to study the JEV 12kDa protein in the context of host immunity?

Studying the JEV 12kDa capsid protein in the context of host immunity requires careful experimental design and consideration of several methodological factors:

• T Cell Response Assessment Protocol:

  • Epitope mapping approaches:

    • Overlapping peptide libraries covering the entire 12kDa sequence

    • MHC binding prediction algorithms to identify potential epitopes

    • Ex vivo stimulation of T cells from infected or vaccinated subjects

  • Functional T cell assays:

    • ELISpot for cytokine-secreting cells

    • Intracellular cytokine staining

    • Proliferation assays

    • Cytotoxicity assays for CD8+ T cells

• B Cell and Antibody Response Methodology:

  • Antibody profiling:

    • Isotype and subclass determination

    • Epitope mapping using peptide arrays or phage display

    • Affinity maturation analysis

  • Neutralization assessment:

    • Plaque reduction neutralization tests

    • Reporter virus neutralization assays

    • Mechanism of neutralization studies

• Innate Immune Response Experimental Design:

  • Pattern recognition receptor (PRR) activation studies:

    • Reporter cell lines for specific PRRs

    • Cytokine profiling in various immune cell types

    • Signaling pathway activation assessment

  • Trained immunity considerations:

    • Epigenetic modifications

    • Metabolic reprogramming

    • Long-term functional changes

• Host-Specific Immune Response Variations:

  • Comparative studies across species:

    • Human vs. mouse vs. pig immune responses

    • Primary cells vs. cell lines

  • Genetic background considerations:

    • MHC haplotype influences

    • Strain-specific variations in animal models

• Immunopathology Assessment Methods:

  • Distinction between protective and pathological immune responses:

    • Cytokine profiling in different disease outcomes

    • Immune cell phenotyping in severe vs. mild disease

    • Correlation with clinical parameters

Studies have shown that JEV activates macrophages via CLEC5A, leading to inflammatory cytokine release, and blockade of this receptor can reduce bystander neuronal damage and JEV-induced proinflammatory cytokine secretion . Similar methodological approaches could be applied to investigate how the 12kDa capsid protein specifically might interact with immune components and contribute to either protective immunity or immunopathology.

What experimental controls are essential when studying post-translational modifications of the JEV 12kDa protein?

Investigation of post-translational modifications (PTMs) on the JEV 12kDa capsid protein requires rigorous experimental controls:

• Expression System Considerations:

  • Comparison of E. coli-expressed protein (lacking eukaryotic PTMs) with:

    • Mammalian cell-expressed protein (HEK293 cells)

    • Insect cell-expressed protein

    • Protein derived from viral infection

  • Controls to account for expression system-specific modifications

• Mass Spectrometry Analysis Controls:

  • Sample preparation controls:

    • Unmodified recombinant protein standards

    • Isotopically labeled internal standards

    • Spiked-in control proteins with known modifications

  • Technical controls:

    • Multiple proteases for comprehensive sequence coverage

    • Both bottom-up and top-down proteomics approaches

    • Multiple fragmentation methods (CID, ETD, HCD)

• Site-Specific Modification Validation Protocol:

  • Site-directed mutagenesis controls:

    • Mutation of putative modification sites to non-modifiable residues

    • Conservative vs. non-conservative substitutions

    • Complete deletion controls

  • Enzymatic modification/demodification:

    • Treatment with specific enzymes (phosphatases, deacetylases, etc.)

    • Enzyme inhibitor controls

    • Time-course analyses

• Functional Significance Assessment Controls:

  • Physiological relevance controls:

    • Comparison of modification states during different infection stages

    • Correlation with viral fitness parameters

    • Host cell type-specific modification patterns

  • Modification-mimicking mutants:

    • Phosphomimetic mutations (e.g., Ser to Asp/Glu)

    • Non-phosphorylatable mutations (e.g., Ser to Ala)

    • Comparison with wild-type protein in functional assays

• Antibody-Based Detection Controls:

  • Specificity controls:

    • Peptide competition assays

    • Use of modification-specific antibodies

    • Validation using modified and unmodified recombinant proteins

  • Signal validation:

    • Secondary antibody-only controls

    • Isotype controls

    • Sequential probing with modification-specific and total protein antibodies

The native JEV envelope protein has been noted to possess all post-translational modifications when expressed in HEK293 cells, delivering optimal antigenicity due to its human origin . Similar considerations should be applied when studying the capsid protein's post-translational modifications, as these may significantly impact function, localization, and immunogenicity.

What are the optimal conditions for structural studies of the JEV 12kDa protein?

Optimizing conditions for structural studies of the JEV 12kDa capsid protein requires careful consideration of multiple parameters:

• Protein Production Optimization Protocol:

  • Expression system selection:

    • E. coli for high yield but potential folding issues

    • HEK293 cells for native folding and post-translational modifications

    • Insect cells as intermediate option

  • Purification strategy:

    • Affinity chromatography with His-tag

    • Size exclusion chromatography for oligomeric state separation

    • Ion exchange chromatography for charge variants separation

  • Quality control metrics:

    • 95% purity by SDS-PAGE

    • Homogeneity assessment by dynamic light scattering

    • Functional activity verification

• Buffer Optimization for Different Structural Methods:

  • X-ray crystallography:

    • Screening of precipitants, buffers, pH, salt concentrations

    • Additives to improve crystal quality

    • Cryoprotection optimization

  • NMR spectroscopy:

    • Buffer compatibility with long acquisition times

    • Deuteration strategies for larger proteins

    • Temperature optimization

  • Cryo-EM:

    • Grid preparation optimization

    • Vitrification conditions

    • Sample concentration adjustments

• Stabilization Strategies:

  • Addition of binding partners:

    • Viral RNA fragments

    • Host factor binding domains

    • Antibody fragments (Fab, scFv)

  • Protein engineering approaches:

    • Surface entropy reduction

    • Disulfide engineering

    • Thermostabilizing mutations

• Data Collection and Processing Considerations:

  • X-ray diffraction:

    • Resolution optimization

    • Radiation damage mitigation

    • Multiple crystal averaging

  • NMR data acquisition:

    • Pulse sequence optimization

    • Relaxation measurements

    • Residual dipolar coupling

  • Cryo-EM:

    • Particle picking strategies

    • Classification approaches

    • Resolution enhancement techniques

• Validation Methodology:

  • Multiple technique validation:

    • Low-resolution techniques (SAXS, negative-stain EM)

    • Complementary structural methods

    • Functional validation of structural insights

The recombinant JEV capsid protein can be stored in PBS with 25mM K₂CO₃ , but buffer optimization for specific structural techniques would likely be required. Long-term storage with carrier proteins (0.1% HSA or BSA) and avoiding freeze-thaw cycles would help maintain sample integrity for structural studies.

How can systems biology approaches be applied to study the JEV 12kDa protein's role in viral-host interactions?

Systems biology offers powerful approaches to comprehensively analyze the JEV 12kDa capsid protein's role in viral-host interactions:

• Multi-omics Integration Methodology:

  • Transcriptomics:

    • RNA-seq of infected vs. uninfected cells

    • Single-cell RNA-seq to capture cellular heterogeneity

    • Temporal profiling during infection progression

  • Proteomics:

    • Global proteome changes upon expression of capsid protein

    • Phosphoproteomics to identify signaling pathway alterations

    • Protein-protein interaction network mapping

  • Metabolomics:

    • Metabolic changes induced by capsid protein expression

    • Flux analysis to identify altered metabolic pathways

    • Integration with proteome data

• Network Analysis Protocol:

  • Protein-protein interaction networks:

    • Identification of capsid protein interactors

    • Network perturbation analysis

    • Hub proteins and critical nodes determination

  • Pathway enrichment analysis:

    • Identification of cellular processes affected by capsid protein

    • Cross-talk between signaling pathways

    • Feedback and feed-forward loops

• Computational Modeling Approaches:

  • Mathematical modeling of infection dynamics

  • Agent-based modeling of capsid protein interactions

  • Prediction of emergent properties and system behavior

  • In silico perturbation studies to guide experimental design

• Integrative Visualization Methods:

  • Multi-dimensional data visualization

  • Temporal mapping of system changes

  • Network visualization tools

  • Interactive dashboards for data exploration

• Validation Strategy:

  • Targeted validation of key network predictions:

    • CRISPR/Cas9 knockout or knockdown studies

    • Overexpression studies

    • Small molecule inhibitors of specific pathways

  • Iterative model refinement:

    • Incorporation of new experimental data

    • Model parameter optimization

    • Prediction-validation cycles

Research has shown that JEV infection activates DAP12 phosphorylation in macrophages , representing one signaling node in a complex network. Systems biology approaches would allow researchers to place this and other capsid protein-mediated events in the context of global cellular response networks, potentially identifying unexpected connections and therapeutic targets.

What novel approaches can be used to study the dynamics of JEV 12kDa protein interactions during the viral lifecycle?

Advanced methodologies to study the dynamics of JEV 12kDa capsid protein interactions throughout the viral lifecycle include:

• Live-Cell Imaging Techniques Protocol:

  • Fluorescent protein tagging strategies:

    • Split-GFP complementation for interaction studies

    • Photoactivatable or photoswitchable fluorescent proteins

    • FRET/FLIM for proximity detection

  • Advanced microscopy methods:

    • Super-resolution microscopy (STORM, PALM, STED)

    • Lattice light-sheet microscopy for 3D dynamics

    • Single-particle tracking

  • Quantitative analysis approaches:

    • Particle tracking and motion analysis

    • Colocalization coefficients

    • Intensity correlation analysis

• Time-Resolved Structural Analysis Methods:

  • Time-resolved X-ray crystallography

  • Time-resolved cryo-EM

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

  • NMR relaxation dispersion experiments

• Temporal Interactome Mapping Strategy:

  • Pulse-chase proteomics

  • BioID or APEX proximity labeling with temporal control

  • Sequential co-immunoprecipitation at defined infection stages

  • Quantitative interaction proteomics with stable isotope labeling

• Correlative Microscopy Approaches:

  • Correlative light and electron microscopy (CLEM)

  • Correlative light and X-ray microscopy

  • Correlative fluorescence and cryo-EM

  • Multi-modal imaging integration

• Real-Time Biosensor Development:

  • FRET-based interaction sensors

  • Split luciferase complementation assays

  • Bioluminescence resonance energy transfer (BRET)

  • Fluorescent RNA aptamers for RNA-protein interaction dynamics

• Experimental Design for Dynamic Studies:

  • Synchronized infection protocols

  • Inducible expression systems

  • Optogenetic control of protein function

  • Microfluidic systems for precise temporal control

The capsid protein plays a critical role in nucleocapsid formation by binding to viral RNA . These advanced approaches would allow researchers to visualize and quantify the dynamics of this process in real-time, as well as to identify transient interactions with host factors that may be missed by static analyses.

How can machine learning approaches be applied to predict epitopes and functional domains of the JEV 12kDa protein?

Machine learning offers powerful tools for predicting epitopes and functional domains within the JEV 12kDa capsid protein:

• Epitope Prediction Methodological Framework:

  • B-cell epitope prediction:

    • Feature extraction from protein sequence and structure

    • Classification algorithms (SVM, Random Forest, Neural Networks)

    • Ensemble methods combining multiple predictors

    • Validation using experimental epitope mapping data

  • T-cell epitope prediction:

    • MHC binding affinity prediction

    • Proteasomal cleavage site prediction

    • TAP transport efficiency modeling

    • Integrated epitope prediction pipelines

• Functional Domain Prediction Protocol:

  • Sequence-based approaches:

    • Evolutionary conservation analysis

    • Motif identification

    • Disorder prediction

    • Secondary structure prediction

  • Structure-based methods:

    • Binding pocket identification

    • Electrostatic surface analysis

    • Molecular dynamics simulation analysis

    • Structural comparison with homologous proteins

• Deep Learning Implementation Strategy:

  • Convolutional neural networks for sequence pattern recognition

  • Recurrent neural networks for capturing sequential dependencies

  • Graph neural networks for structural data

  • Attention mechanisms for identifying critical regions

  • Transfer learning from related viral proteins

• Integrative Prediction Approaches:

  • Multi-modal data integration:

    • Sequence, structure, dynamics, and evolutionary data

    • Experimental binding data

    • Literature-derived information

  • Multi-task learning:

    • Simultaneous prediction of multiple properties

    • Parameter sharing across related prediction tasks

• Experimental Validation Design:

  • Targeted mutagenesis of predicted sites

  • Peptide binding assays for epitope validation

  • Functional assays for domain verification

  • Structural studies of predicted interaction sites

Machine learning approaches could be particularly valuable for the JEV capsid protein, where understanding of specific functional domains and epitopes remains limited. The recombinant JEV proteins that have been produced for research purposes provide valuable experimental data that could be used to train and validate these computational models.

What are the key methodological considerations for researchers beginning work with the JEV 12kDa protein?

Researchers initiating studies on the JEV 12kDa capsid protein should consider several critical methodological factors:

• Protein Source and Production Considerations:

  • Expression system selection based on research goals:

    • E. coli for high-yield, cost-effective production (110 amino acids, 12kDa)

    • Mammalian cells (HEK293) for native folding and post-translational modifications

  • Purification strategy optimization:

    • His-tag fusion for affinity purification

    • Chromatographic techniques to achieve >95% purity

    • Quality control by SDS-PAGE and functional assays

• Storage and Handling Protocol:

  • Buffer selection (PBS with 25mM K₂CO₃ has been used successfully)

  • Temperature considerations:

    • 4°C for short-term storage (2-4 weeks)

    • -20°C with carrier proteins (0.1% HSA or BSA) for long-term storage

  • Avoidance of multiple freeze-thaw cycles

  • Aliquoting strategy for experimental use

• Experimental Design Fundamentals:

  • Appropriate controls:

    • Unrelated proteins of similar size

    • Denatured protein controls

    • Host cell-derived control proteins

  • Concentration optimization for specific applications

  • Validation of protein functionality before complex experiments

• Safety Considerations:

  • Although recombinant protein lacks infectivity, proper laboratory safety protocols

  • Consideration of biosafety levels when working with infectious JEV

  • Proper decontamination procedures

• Application-Specific Methodological Considerations:

  • ELISA applications: Optimization of coating conditions, blocking, and detection

  • Structural studies: Buffer optimization, stability assessment

  • Cell-based assays: Endotoxin testing, delivery method optimization

  • Animal studies: Formulation with appropriate adjuvants

• Data Analysis and Reporting Standards:

  • Proper statistical approach based on experimental design

  • Transparent reporting of methods and materials

  • Data sharing according to field standards

Understanding that the 12kDa capsid protein binds to viral RNA to form a nucleocapsid that is enveloped by an endoplasmic reticulum-derived membrane containing E and prM proteins provides important context for experimental design. Researchers should also consider the potential role of the protein in neuroinflammation, as JEV infection has been shown to trigger inflammatory responses through mechanisms involving host receptors like CLEC5A .

How can researchers integrate findings about the JEV 12kDa protein into broader understanding of flavivirus biology?

Integrating research findings on the JEV 12kDa capsid protein into the broader context of flavivirus biology requires systematic methodological approaches:

• Comparative Analysis Framework:

  • Cross-species protein comparison:

    • Sequence and structural alignment with homologous proteins from related flaviviruses

    • Identification of conserved vs. variable regions

    • Correlation with host range and tissue tropism

  • Functional conservation assessment:

    • RNA binding properties

    • Protein-protein interaction networks

    • Subcellular localization patterns

• Integrated Data Analysis Protocol:

  • Meta-analysis of published literature:

    • Systematic review methodology

    • Quantitative synthesis of experimental results

    • Identification of consistent findings vs. conflicting data

  • Database integration:

    • Incorporation of findings into virus-specific databases

    • Contribution to broader protein interaction databases

    • Structural data deposition

• Translational Research Methodology:

  • Application to vaccine development:

    • Incorporation of findings about immunogenic epitopes

    • Design of next-generation vaccines based on capsid protein insights

    • Correlates of protection identification

  • Antiviral development:

    • Target identification based on essential functions

    • Rational drug design informed by structural data

    • Development of broad-spectrum antivirals targeting conserved features

• Collaborative Research Strategies:

  • Multi-laboratory validation studies

  • Standardization of protocols for cross-comparison

  • Sharing of reagents and resources

  • Interdisciplinary approaches combining virology, immunology, and structural biology

• Knowledge Dissemination Plan:

  • Development of accessible databases and visualization tools

  • Educational resources for researchers entering the field

  • Regular review articles synthesizing current understanding