Dengue Envelope-3, Insect

What is the structure and function of dengue virus envelope protein domain III?

Domain III (EDIII) of the dengue virus envelope (E) protein is a crucial structural component located at the C-terminal end of the E protein. Structurally, EDIII adopts an immunoglobulin-like fold and projects from the virion surface. It functions primarily as the receptor-binding domain, mediating virus attachment to host cells during infection . The domain consists of approximately 100 amino acids and contains both type-specific and cross-reactive epitopes that are recognized by neutralizing antibodies .

Research methodologies to study EDIII structure include:

• X-ray crystallography to determine three-dimensional structure

• Recombinant protein expression systems (bacterial, yeast, or baculovirus)

• Surface plasmon resonance for binding affinity measurements

• Epitope mapping using alanine scanning mutagenesis approaches

How does EDIII differ across the four dengue virus serotypes?

While EDIII maintains a conserved structural fold across all four dengue virus serotypes (DENV-1 to DENV-4), it exhibits significant sequence variability that contributes to serotype specificity. This variability primarily occurs in surface-exposed loops, particularly the lateral ridge consisting of the BC, DE, and FG loops .

Methodological approaches to analyze serotype differences include:

• Sequence alignment analysis to identify variable and conserved regions

• Epitope mapping studies using monoclonal antibodies

• Structural comparison through superimposition of crystal structures

• Functional binding assays with host receptors

• Neutralization assays using serotype-specific antibodies

The lateral ridge region of EDIII, particularly centered around residues E383 and P384 in DENV-2, has been identified as containing serotype-specific epitopes that elicit type-specific neutralizing antibodies .

What are the key insect proteins that interact with dengue virus EDIII?

Several mosquito proteins interact with dengue virus EDIII during the infection cycle. One significant interaction partner is lachesin, a neuronal cell surface protein in Aedes aegypti that directly binds to EDIII . Through phage display library screening and protein interaction studies, lachesin has been shown to be important for DENV replication in mosquito tissues .

To identify and characterize such interactions, researchers employ:

• Phage display library screening against EDIII

• Co-immunoprecipitation assays

• Yeast two-hybrid screening

• Protein-protein interaction assays with purified components

• RNA interference (RNAi) to validate functional significance

• Immunofluorescence microscopy for colocalization studies

Blocking lachesin protein with specific antibodies significantly reduces DENV replication in mosquitoes, highlighting its importance in the virus life cycle .

How do researchers express and purify recombinant EDIII for experimental use?

Various expression systems have been employed to produce recombinant EDIII proteins for research applications. The methodological approaches include:

• Baculovirus Expression System:

  • Advantages: Produces secreted protein with preserved native conformation

  • Yield: Approximately 300μg per 10^6 infected Sf9 cells

  • Purification: Immobilized affinity chromatography

  • Quality control: SDS-PAGE for purity assessment and Western blot for reactivity

• Pichia pastoris Expression:

  • Advantages: Cost-effective and scalable yeast-based system

  • Applications: Useful for immunization studies

  • Post-translational modifications: Can include certain eukaryotic modifications

• E. coli Expression Systems:

  • Advantages: High yield and simplicity

  • Limitations: May require refolding for proper conformation

  • Purification: Typically employs affinity tags (His-tag, GST, etc.)

Each system presents different advantages for specific applications, with the choice depending on the intended use (structural studies, diagnostics, or immunization) and the requirement for post-translational modifications.

What are the molecular mechanisms of EDIII-mediated entry into mosquito cells?

The entry of dengue virus into mosquito cells involves complex molecular interactions between viral EDIII and multiple mosquito cellular components. Recent research has revealed that:

• EDIII interacts with specific mosquito cell receptors, with lachesin identified as a key neuronal binding partner .

• The virus traverses through multiple mosquito tissues during the transmission cycle:

  • Initial infection occurs in the midgut after blood meal

  • Virus disseminates through hemolymph to secondary tissues

  • Final replication in salivary glands precedes transmission

• Dengue virus enhances midgut permeability by capturing human plasmin from the blood meal, allowing for more efficient infection .

Experimental approaches to study these mechanisms include:

• Ex vivo mosquito tissue binding assays

• Immunofluorescence microscopy to track viral progression

• Transmission electron microscopy for ultrastructural analysis

• RNA interference to silence potential receptor genes

• Transgenic mosquito models with labeled cellular components

• Antibody blocking experiments targeting specific interactions

How can epitope mapping of EDIII inform the development of serotype-specific diagnostics?

Epitope mapping of EDIII has significant implications for developing serotype-specific diagnostics. Methodological approaches include:

• High-throughput epitope mapping techniques:

  • Alanine scanning mutagenesis creating libraries of mutants

  • Dot blot assays using 67+ alanine mutants of surface-exposed E residues

  • Capture-ELISA confirmation assays

  • Phage display peptide libraries

• Identification of serotype-specific epitopes:

  • Type-specific neutralizing epitopes for DENV-2 localize to the lateral ridge of EDIII

  • Centered at the FG loop near residues E383 and P384

  • Adjacent epitopes on the connecting A strand at residues K305, K307, and K310 are recognized by subcomplex-specific antibodies

  • Cross-reactive epitopes often map to the AB loop

• Translation to diagnostic applications:

  • Using recombinant EDIII from multiple serotypes improves diagnostic sensitivity

  • Combined results from DENV-1, DENV-3, and DENV-4 rEDIII-ELISA achieved 81.82% sensitivity and 100% specificity for acute phase infection

  • Each serotype's EDIII can compensate for negative results from other serotypes

This knowledge enables the rational design of diagnostic assays with enhanced performance for early detection of dengue infection.

What strategies can overcome the limitations of EDIII-based vaccines regarding cross-protection?

EDIII-based vaccines face challenges in providing cross-protection against all four dengue serotypes. Advanced strategies to address these limitations include:

• Tetravalent EDIII constructs:

  • Connecting EDIII from all four serotypes in a single protein (e.g., D1-D3-D4-D2 arrangement)

  • Expression in baculovirus systems to maintain native conformation

  • Ensuring stability and proper presentation of all four domains

• Epitope-focused immunogen design:

  • Engineering N-glycosylation sites on EDIII to shield non-neutralizing epitopes

  • Directing the immune response toward potently neutralizing epitopes

  • Minimizing antibody-dependent enhancement (ADE) potential

  • Example: EDIII mutant N with glycosylation sites that selectively display neutralizing epitopes

• Prime-boost strategies:

  • Sequential immunization with different serotypes

  • Combined immunization with tetravalent constructs

  • Analysis of antibody responses to identify optimal protocols

• Addressing limitations of current approaches:

  • Recognition that EDIII immunization may elicit cross-reactive antibodies to the conserved AB loop with poor neutralizing activity

  • Understanding that some epitopes are less accessible on the mature virion surface

  • Developing strategies to target interdomain epitopes that may be more accessible

Experimental assessment of these strategies requires comprehensive neutralization assays against all serotypes and evaluation of ADE potential in vitro and in animal models.

How do insect-specific factors influence the evolution of EDIII sequences?

The co-evolution of dengue virus EDIII and its mosquito host factors represents an intricate area of research. Methodological approaches to understand this relationship include:

• Comparative genomics and selective pressure analysis:

  • Sequencing EDIII from dengue viruses isolated from different geographic regions with distinct vector populations

  • Calculating non-synonymous to synonymous substitution ratios (dN/dS) to identify sites under positive selection

  • Comparing EDIII sequences from human versus mosquito isolates to identify vector-specific adaptations

• Experimental evolution studies:

  • Serial passage of dengue virus in mosquito cells versus mammalian cells

  • Deep sequencing to identify adaptive mutations in EDIII

  • Fitness competition assays between wild-type and mutant viruses

• Vector competence studies:

  • Assessing how EDIII variations affect virus transmission efficiency

  • Examining infection, dissemination, and transmission rates in different mosquito populations

This research provides insights into how selective pressures from insect hosts shape EDIII sequence diversity and function, with implications for viral adaptation and transmission dynamics.

What are the methodological challenges in studying EDIII-antibody interactions at the molecular level?

Investigating EDIII-antibody interactions at the molecular level presents several methodological challenges that researchers must address:

• Structural analysis challenges:

  • Obtaining high-resolution co-crystal structures of EDIII with antibodies

  • Distinguishing interdomain epitopes that may span multiple domains

  • Capturing dynamic aspects of antibody binding

• Epitope mapping complexities:

  • Differentiating between continuous versus discontinuous epitopes

  • Identifying epitopes that involve quaternary structures on intact virions

  • Resolving epitopes recognized by polyclonal sera

  • Addressing the frequent presence of interdomain epitopes previously underappreciated

• Functional correlation difficulties:

  • Connecting binding affinity with neutralization potential

  • Understanding why some epitopes elicit potent neutralization while others do not

  • Reconciling data from different neutralization assay formats

  • Accounting for differences in epitope accessibility on various forms of the virus (immature, mature, and partially mature virions)

• Methodological approaches to address these challenges:

  • Cryo-electron microscopy of virion-Fab complexes

  • Hydrogen-deuterium exchange mass spectrometry

  • Single-molecule fluorescence resonance energy transfer (FRET)

  • Surface plasmon resonance with kinetic analysis

  • Yeast surface display for fine epitope mapping

  • High-throughput mutational scanning combined with deep sequencing

Understanding these interactions is crucial for rational vaccine design and therapeutic antibody development.

How should researchers design experiments to evaluate EDIII-based diagnostic tools for field applications?

Designing robust evaluations of EDIII-based diagnostic tools requires careful experimental planning:

• Sample selection and characterization:

  • Include well-characterized panels of sera from confirmed dengue cases (all serotypes)

  • Include samples from different stages of infection (acute, convalescent)

  • Include relevant controls (other flavivirus infections, non-flavivirus febrile illnesses, healthy controls)

  • Ensure geographic diversity of samples to account for strain variations

• Performance assessment metrics:

  • Sensitivity and specificity calculations with confidence intervals

  • Cross-reactivity analysis with other flaviviruses

  • Detection limits and dynamic range determination

  • Reproducibility assessment (intra- and inter-assay variability)

• Field-relevant conditions testing:

  • Stability testing under various temperature and humidity conditions

  • Shelf-life determination

  • Performance with minimally processed samples

  • Usability in resource-limited settings

• Validation against gold standard methods:

  • Comparison with virus isolation

  • Correlation with RT-PCR results

  • Agreement with established serological methods

A well-designed MAC-ELISA based on tetravalent recombinant EDIII has demonstrated 93% sensitivity and 100% specificity , while combining results from multiple serotype-specific EDIII-ELISAs achieved 81.82% sensitivity and 100% specificity for acute phase detection .

What control experiments are essential when studying EDIII interactions with mosquito proteins?

When investigating interactions between dengue EDIII and mosquito proteins, several critical control experiments must be included:

• Protein-protein interaction controls:

  • Positive controls: Known interacting protein pairs

  • Negative controls: Unrelated proteins unlikely to interact

  • Competition assays with soluble proteins to demonstrate specificity

  • Dose-response binding curves to assess affinity

• Functional validation controls:

  • RNAi knockdown controls (non-targeting siRNA/shRNA)

  • Phenotype rescue experiments with RNAi-resistant constructs

  • Antibody specificity validation for blocking experiments

  • Mock infection controls alongside experimental infections

• Localization studies controls:

  • Secondary antibody-only controls for immunofluorescence

  • Counterstaining with organelle markers

  • Confocal z-stack analysis to confirm true colocalization

  • Live cell imaging controls to rule out fixation artifacts

• Binding specificity controls:

  • Testing interactions with all four DENV serotypes

  • Using related flavivirus proteins as specificity controls

  • Domain deletion mutants to map interaction interfaces

  • Site-directed mutagenesis of key residues

These controls are exemplified in studies of lachesin-EDIII interactions, where phage display screening was followed by cloning, expression, purification, and in vitro interaction studies, with validation through confocal microscopy and antibody blocking experiments .

How can researchers design experiments to distinguish type-specific from cross-reactive antibody responses to EDIII?

Designing experiments to differentiate type-specific from cross-reactive antibody responses requires sophisticated methodological approaches:

• Antigen preparation strategies:

  • Express recombinant EDIII from all four dengue serotypes

  • Create chimeric EDIII constructs with swapped epitope regions

  • Generate alanine substitution mutants at key epitope residues

  • Develop conformational versus denatured EDIII antigens

• Competitive binding assays:

  • Pre-absorption of sera with heterologous EDIII proteins

  • Sequential binding assays with different serotypes

  • Epitope-blocking experiments with characterized monoclonal antibodies

  • Competition ELISA between labeled and unlabeled antibodies

• Functional differentiation methods:

  • Serotype-specific neutralization assays

  • Antibody-dependent enhancement assays

  • Avidity measurements for different serotypes

  • Isotype and subclass profiling of the antibody response

• Advanced analytical approaches:

  • Depletion studies removing cross-reactive antibodies

  • Single B-cell isolation and monoclonal antibody generation

  • Deep sequencing of antibody repertoires

  • Structural analysis of antibody-antigen complexes

These approaches have revealed that type-specific neutralizing antibodies target the lateral ridge of EDIII (particularly the FG loop), while cross-reactive antibodies with poor neutralizing activity often recognize the conserved AB loop region .

What experimental approaches can assess the impact of EDIII mutations on mosquito vector competence?

Evaluating how EDIII mutations affect mosquito vector competence requires a comprehensive experimental framework:

• Engineering viral EDIII variants:

  • Site-directed mutagenesis of infectious cDNA clones

  • Reverse genetics to generate recombinant viruses

  • Creation of chimeric viruses with swapped EDIII regions

  • CRISPR-Cas9 editing of viral genomes

• Vector competence assessment protocols:

  • Controlled mosquito infection through membrane feeding

  • Measurement of infection rates in midgut tissues

  • Quantification of dissemination to secondary tissues

  • Determination of transmission efficiency through forced salivation

  • Viral load quantification by RT-qPCR or plaque assays

• Mechanistic investigations:

  • Binding assays with mosquito tissue extracts

  • Immunohistochemistry to track viral progression

  • Ex vivo infection of isolated mosquito tissues

  • Transcriptomic analysis of mosquito response to different viral variants

• Field-relevant conditions:

  • Testing at different temperatures to mimic environmental variation

  • Using field-derived mosquito populations

  • Including competitive infection experiments with wild-type virus

These approaches can reveal how specific mutations in EDIII, particularly in regions that interact with mosquito proteins like lachesin, affect the virus's ability to infect and be transmitted by mosquito vectors.

How should researchers interpret discrepancies between in vitro binding data and in vivo protection for EDIII-targeting antibodies?

Interpreting discrepancies between in vitro binding and in vivo protection requires careful consideration of multiple factors:

• Epitope accessibility considerations:

  • EDIII epitopes may have different accessibility on virions versus recombinant proteins

  • Some epitopes (like the AB loop) show strong binding in vitro but are poorly accessible on mature virions

  • Epitopes may be differentially exposed during various stages of the viral life cycle

• Functional antibody properties beyond binding:

  • Antibody affinity versus avidity effects

  • Isotype-dependent effector functions

  • Ability to cross the endosomal membrane

  • Timing of neutralization (pre- or post-attachment)

• Statistical approaches for analysis:

  • Correlation analysis between binding parameters and protection

  • Multivariate modeling to identify predictive factors

  • Receiver operating characteristic (ROC) curves to determine predictive thresholds

  • Principal component analysis to identify patterns in complex datasets

• Strategies to reconcile discrepancies:

  • Combined in vitro assays that better mimic in vivo conditions

  • Pre-and post-attachment neutralization assays

  • Using virions at different maturation states

  • Testing antibodies against diverse viral strains

Understanding these discrepancies is critical, as demonstrated by studies showing that EDIII immunization can elicit antibodies to conserved epitopes that bind strongly in vitro but contribute inefficiently to neutralization due to limited exposure on the virion surface .

What statistical approaches are most appropriate for analyzing epitope mapping data from EDIII mutant panels?

Analyzing epitope mapping data from EDIII mutant panels requires sophisticated statistical approaches:

• Data normalization methods:

  • Percent binding relative to wild-type

  • Z-score normalization across mutant panels

  • Internal reference controls for inter-assay normalization

  • Background subtraction algorithms

• Classification of binding phenotypes:

  • Hierarchical clustering of mutant binding profiles

  • Principal component analysis to identify epitope patterns

  • Machine learning approaches for epitope prediction

  • Network analysis of residue interactions

• Statistical significance testing:

  • Multiple testing correction (Bonferroni, False Discovery Rate)

  • ANOVA with post-hoc tests for multiple comparisons

  • Non-parametric methods for non-normally distributed data

  • Bootstrap resampling for confidence interval estimation

• Visualization techniques:

  • Heat maps of binding reduction across mutant panels

  • Structural mapping of significant residues

  • Epitope fingerprinting diagrams

  • Three-dimensional visualization of binding footprints

These approaches were exemplified in a high-throughput dot blot assay using 67 alanine mutants of predicted surface-exposed E residues, revealing novel epitopes at the central interface of domain II and interdomain epitopes spanning domains II and III .

How can researchers resolve contradictions in the literature regarding EDIII-based vaccine efficacy?

Resolving contradictions in EDIII-based vaccine efficacy literature requires systematic approaches:

• Meta-analytical techniques:

  • Systematic literature review with inclusion/exclusion criteria

  • Standardized data extraction protocols

  • Effect size calculations across studies

  • Heterogeneity assessment (I² statistic)

  • Publication bias evaluation (funnel plots, Egger's test)

• Identifying sources of variation:

  • Differences in immunization protocols (dose, schedule, adjuvants)

  • Variation in challenge models and strains

  • Diverse immunological readouts and endpoints

  • Host factors (genetic background, age, previous exposure)

  • Antigen design differences (sequence, structure, glycosylation)

• Reconciliation strategies:

  • Direct head-to-head comparisons under standardized conditions

  • Immune correlate analysis across studies

  • Mechanistic studies to explain divergent outcomes

  • Individual participant data meta-analysis

• Design considerations for future studies:

  • Standardized reporting frameworks (ARRIVE guidelines)

  • Pre-registration of study protocols

  • Multi-laboratory validation studies

  • More comprehensive immunological assessment

This approach has revealed that factors like epitope accessibility explain why EDIII immunization may elicit strong antibody responses that do not translate to protection, as the targeted epitopes may be poorly exposed on the virion surface .

What are the most reliable methods to quantify EDIII binding to insect cell receptors?

Quantifying EDIII binding to insect cell receptors requires reliable methodological approaches:

• In vitro binding assays:

  • Surface plasmon resonance (SPR) for real-time kinetic measurements

  • Bio-layer interferometry for label-free interaction analysis

  • ELISA-based binding assays with purified receptors

  • Flow cytometry with fluorescently labeled EDIII

  • Microscale thermophoresis for solution-phase interactions

• Cellular binding studies:

  • Cell-based ELISA with fixed insect cells

  • Competitive binding assays with labeled/unlabeled EDIII

  • FACS analysis of EDIII binding to intact cells

  • Confocal microscopy with quantitative image analysis

  • Live cell imaging with fluorescently tagged proteins

• Data analysis considerations:

  • Saturation binding curves with nonlinear regression

  • Scatchard plot analysis for multiple binding sites

  • Competitive binding analysis using Cheng-Prusoff equation

  • Statistical comparison of binding parameters (Kd, Bmax)

• Validation approaches:

  • Multiple independent methods showing consistent results

  • Positive and negative control proteins

  • Dose-dependent inhibition with competing ligands

  • Genetic manipulation of receptor expression levels

These methods have been applied to study interactions between DENV EDIII and insect proteins like lachesin, demonstrating specific binding and functional relevance through antibody blocking experiments that reduced viral replication .

How might advances in structural biology techniques enhance our understanding of EDIII-insect protein interactions?

Emerging structural biology techniques offer unprecedented opportunities to advance our understanding of EDIII-insect protein interactions:

• Cryo-electron microscopy (cryo-EM) applications:

  • Single-particle analysis of EDIII-receptor complexes

  • Tomography of virus-membrane interactions in insect cells

  • Time-resolved structural studies of binding events

  • In situ structural determination within cellular contexts

• Integrative structural biology approaches:

  • Combining X-ray crystallography, NMR, and cryo-EM data

  • Small-angle X-ray scattering (SAXS) for solution structures

  • Mass spectrometry for structural proteomics

  • Computational modeling and molecular dynamics simulations

• Emerging techniques:

  • Microcrystal electron diffraction (MicroED)

  • Cryo-electron tomography with subtomogram averaging

  • Time-resolved serial crystallography at X-ray free-electron lasers

  • Advanced nuclear magnetic resonance spectroscopy methods

• Biological applications:

  • Mapping interaction interfaces between EDIII and lachesin

  • Determining structural basis for serotype specificity

  • Visualizing conformational changes during binding

  • Structural analysis of how plasmin capture by the envelope protein enhances mosquito midgut permeability

These techniques could reveal critical details about how EDIII interacts with mosquito proteins at atomic resolution, providing insights for designing transmission-blocking interventions.

What potential exists for targeting EDIII-insect protein interactions for transmission-blocking strategies?

Targeting EDIII-insect protein interactions presents promising opportunities for transmission-blocking interventions:

• Small molecule inhibitor development:

  • High-throughput screening of compound libraries

  • Structure-based drug design targeting interaction interfaces

  • Fragment-based approaches to identify binding scaffolds

  • Peptidomimetic inhibitors of key protein-protein interactions

• Immunological approaches:

  • Transmission-blocking antibodies targeting EDIII-insect protein interfaces

  • Vaccination strategies eliciting antibodies that disrupt transmission

  • Nanobody/single-domain antibody development

  • Fc-engineered antibodies with enhanced mosquito tissue penetration

• Genetic strategies:

  • CRISPR-mediated modification of insect receptor genes

  • Transgenic mosquitoes expressing interaction-disrupting proteins

  • Population replacement strategies with transmission-resistant mosquitoes

  • RNA interference approaches targeting receptor expression

• Rational design based on mechanistic insights:

  • Targeting the lachesin-EDIII interaction to reduce viral replication

  • Disrupting the mechanism by which dengue virus uses its envelope protein to capture human plasmin and enhance mosquito midgut permeability

  • Developing inhibitors of other mosquito neuronal proteins involved in DENV transmission

Experimental validation would require assessing impact on vector competence, measuring reduction in transmission efficiency, and evaluating evolutionary escape mechanisms.

How can machine learning approaches enhance EDIII epitope prediction and vaccine design?

Machine learning (ML) offers powerful tools for advancing EDIII epitope prediction and vaccine design:

• Advanced epitope prediction algorithms:

  • Deep learning models trained on existing epitope mapping data

  • Convolutional neural networks for structure-based epitope prediction

  • Recurrent neural networks for sequence-based analysis

  • Ensemble methods combining multiple prediction approaches

• Immunogen design applications:

  • Generative adversarial networks for novel immunogen design

  • Reinforcement learning to optimize epitope presentation

  • ML-guided glycosylation site prediction to shield non-neutralizing epitopes

  • Neural network models to predict immunogenicity

• Implementation methodologies:

  • Transfer learning from related flavivirus datasets

  • Unsupervised clustering to identify epitope patterns

  • Active learning approaches to guide experimental design

  • Interpretable ML to understand epitope determinants

• Experimental validation frameworks:

  • Iterative design-build-test cycles guided by ML predictions

  • High-throughput experimental validation of ML-predicted epitopes

  • Integration with structural biology data

  • In silico prediction followed by targeted mutagenesis

These approaches could advance epitope-focused vaccine design strategies, such as the N-glycosylated EDIII antigen that selectively displays neutralizing epitopes while shielding others to drive selection of potently neutralizing antibodies with minimal enhancement potential .

What are the implications of viral evolution and quasispecies dynamics for EDIII-based interventions?

Understanding viral evolution and quasispecies dynamics has critical implications for EDIII-based interventions:

• Methodological approaches to study EDIII evolution:

  • Next-generation sequencing of viral populations

  • Deep mutational scanning of EDIII tolerance to mutations

  • Ancestral sequence reconstruction

  • Bayesian evolutionary analysis

  • Selection pressure mapping (dN/dS ratios)

• Key considerations for intervention design:

  • Identifying evolutionary constraints in EDIII

  • Targeting functionally critical, evolutionarily conserved epitopes

  • Predicting potential escape mutations

  • Designing combinatorial approaches to mitigate resistance

  • Understanding fitness costs of escape mutations

• Experimental systems to assess evolutionary dynamics:

  • In vitro selection under antibody pressure

  • Serial passage in the presence of inhibitors

  • Competition assays between wild-type and mutant viruses

  • Mathematical modeling of evolutionary trajectories

• Implications for different intervention types:

  • Multi-target antibody cocktails to prevent escape

  • Structure-based design of broad-spectrum inhibitors

  • Epitope-focused vaccines targeting conserved regions

  • Transmission-blocking strategies targeting essential insect interactions

These considerations are particularly relevant for designing interventions that remain effective despite dengue's genetic diversity and ability to evolve under selective pressure.

How do post-translational modifications of EDIII impact its interactions with insect proteins?

Post-translational modifications (PTMs) of EDIII can significantly affect its interactions with insect proteins:

• Types of PTMs to investigate:

  • N-linked and O-linked glycosylation

  • Phosphorylation

  • Ubiquitination

  • SUMOylation

  • Proteolytic processing

• Methodological approaches for PTM analysis:

  • Mass spectrometry-based proteomics

  • Site-directed mutagenesis of modification sites

  • Expression in different systems with varying PTM capabilities

  • Glycoproteomic analysis

  • Lectin binding assays for glycosylation

• Functional impact assessment:

  • Binding assays comparing modified and unmodified EDIII

  • Cell entry studies with differentially modified proteins

  • Transmission studies in mosquitoes

  • Structural analysis of how PTMs affect protein-protein interfaces

• Applications in intervention design:

  • Engineering N-glycosylation sites to direct immune responses toward specific epitopes

  • Using glycan shielding to mask non-neutralizing epitopes

  • Developing inhibitors that target PTM-dependent interactions

  • Creating transmission-blocking agents that interfere with PTM-mediated processes