TBEV gE C-end

What is the structure of the TBEV envelope glycoprotein and its C-terminal region?

The TBEV envelope (E) glycoprotein consists of an ectodomain (residues 1-396) that forms antiparallel dimers on the virion surface in a "herringbone" pattern with icosahedral symmetry . The E protein structure contains three domains (I, II, and III), followed by a stem region and a transmembrane anchor at the C-terminal end. The C-terminal region includes both the stem and anchor portions, which are often not well-resolved in structural studies, particularly after formaldehyde treatment for virus inactivation . The structure of the E protein has been determined at 3.0 Å resolution using cryo-electron microscopy, revealing the arrangement of three symmetry-independent ectodomain subunits (referred to as E2, E3, and E5) in the asymmetric unit .

How does N-glycosylation affect the TBEV envelope glycoprotein?

N-glycosylation of the TBEV E protein occurs at specific asparagine residues, notably Asn154, and is critical for virus egress in mammalian cells but not in tick cells . Mass spectrometric analysis has revealed significant differences in glycosylation profiles between TBEV grown in human neuronal cells versus tick cells . In human neuronal cells, the main glycan structures include:

• High-mannose glycan with five mannose residues (Man₅GlcNAc₂)

• Complex biantennary galactosylated structure with core fucose (2Gal-2GlcNAc-2Man-3GlcNAc-2Fuc)

• Hybrid glycans with composition Gal₀₋₁GlcNAc₁Man₃₋₅GlcNAc₂Fuc

In contrast, TBEV grown in tick cells primarily shows paucimannose (Man₃₋₄GlcNAc₂Fuc) and high-mannose structures with five and six mannoses . These glycans may mask specific antigenic sites from recognition by neutralizing antibodies, affecting immune responses to the virus.

What methods are most effective for studying the structure of TBEV E protein's C-terminal region?

For structural analysis of TBEV E protein, including its C-terminal region, a combination of techniques has proven effective:

• Cryo-electron microscopy (cryo-EM): This has been the gold standard for determining virion structure at high resolution (3.0 Å), allowing visualization of the E protein ectodomain arrangement . The sample preparation involves formaldehyde inactivation, centrifugation through sucrose density gradients, and resuspension in TNE/5 buffer .

• Computational modeling: Programs like AlphaFold2 have been used to generate initial atomic models of the E protein ectodomain, which can then be fitted into electron density maps using software such as Coot, Phenix, and Isolde .

• Mass spectrometry: For analyzing glycosylation patterns of the E protein in different host cells .

• Structure analysis software: Tools including CCP4 suite, PyMol, Chimera, and ChimeraX allow for detailed structural analysis and visualization . For analyzing accessible surface cavities in the protein structure, the V3 program using rolling probe method has been applied .

It's important to note that while the ectodomain is well-resolved in these analyses, the stem and anchor regions of the E protein (the C-terminal portions) are often not visualized, particularly in formaldehyde-inactivated virus preparations .

What is the significance of the quasispecies variation in the TBEV E protein sequence?

TBEV, like other RNA viruses, exists as quasispecies due to the error-prone nature of its RNA-dependent RNA polymerase, which generates genetically distinct virus variants . Deep sequencing analysis has revealed several non-synonymous mutations in the E protein gene, indicating potential functional significance . These variations may contribute to:

• Adaptation to different hosts (vertebrate and invertebrate)

• Immune evasion strategies

• Changes in virulence or transmission efficiency

How do the structural variations in the C-terminal region of TBEV E protein affect viral pathogenesis across different subtypes?

The C-terminal region of the TBEV E protein, comprising the stem and transmembrane domains, plays crucial roles in viral assembly, membrane fusion, and host cell interaction. Structural variations in this region may contribute to differences in pathogenicity observed among TBEV subtypes (Western/European, Siberian, and Far-Eastern) .

Phylogenetic analysis of over 225 TBEV strains reveals distinct evolutionary patterns across subtypes, with implications for structure-function relationships in the E protein . The TBEVnext dataset (available at https://nextstrain.org/groups/ViennaRNA/TBEVnext) demonstrates that TBEV can be classified into several subtypes and lineages:

• Eastern types: TBEV-FE (Far-Eastern), TBEV-Sib (Siberian)

• Western type: TBEV-Eur (European)

• Smaller lineages: TBEV-Bkl-1, TBEV-Bkl-2 (East-Siberian/Baikalean), Western European, and Himalayan

These subtypes show geographic specificity but can overlap in regions like Irkutsk Oblast in Russia, where multiple subtypes coexist . While the search results don't provide specific data on C-terminal variations across subtypes, the molecular evolution patterns suggest that subtype-specific differences in the E protein, including its C-terminal region, may influence host adaptation and pathogenicity.

Research approaches to address this question would include:

• Comparative structural analysis of E proteins across subtypes

• Mutational studies targeting C-terminal residues

• Host-pathogen interaction assays with chimeric viruses

• Animal models comparing pathogenicity of engineered variants

What experimental approaches are most suitable for analyzing the interaction between TBEV E protein's C-terminal region and host immune responses?

Understanding how the C-terminal region of the TBEV E protein interacts with host immune components requires sophisticated experimental approaches:

• T-cell epitope mapping: Longitudinal studies of TBEV patients have identified HLA-A2-restricted and HLA-B7-restricted CD8+ T cell epitopes . Similar approaches could be applied specifically to the C-terminal region to identify potential epitopes. This involves:

  • PBMC isolation from TBEV patients

  • Tetramer development for tracking virus-specific T cells

  • Phenotypic characterization (CD45RA, CCR7, PD-1, etc.)

  • Functional assays (cytokine production, proliferation)

• Glycosylation impact assessment: Since glycosylation patterns differ between mammalian and tick cells , methodologies to evaluate how these differences affect C-terminal region recognition include:

  • Mass spectrometry analysis of glycopeptides

  • Enzymatic modification of glycans

  • Binding assays with neutralizing antibodies

  • Mutagenesis of glycosylation sites

• Structural immunology approaches: The 3.0 Å resolution cryo-EM structure provides a foundation for mapping antibody binding sites . Techniques include:

  • Antibody-virus complex cryo-EM

  • Computational epitope prediction

  • Hydrogen-deuterium exchange mass spectrometry

  • Surface plasmon resonance for binding kinetics

How do RNA secondary structures in the coding region for TBEV E protein influence protein folding and function?

TBEV genome contains conserved RNA secondary structures that extend beyond the untranslated regions (UTRs) into coding regions, potentially including the E protein gene . These structures may affect:

• Translation efficiency and protein folding

• Genome packaging and assembly

• Evasion of host immune sensors

• Co-evolutionary constraints on amino acid sequences

Research approaches to investigate these relationships include:

• Comparative genomic analysis: Examining conservation patterns across subtypes to identify structurally constrained regions

• Thermodynamic modeling: Predicting RNA secondary structures in the E protein coding region

• SHAPE (Selective 2′-hydroxyl acylation analyzed by primer extension) analysis: Experimentally determining RNA structures in vitro and in cells

• Mutational studies: Introducing synonymous mutations that alter RNA structure but preserve amino acid sequence

• Ribosome profiling: Assessing translation efficiency across the E protein coding region

This type of analysis would complement the current understanding of TBEV UTR structureome diversity that has been characterized through comparative genomic approaches and thermodynamic modeling .

What are the optimal procedures for isolating and purifying TBEV for structural studies of its E protein?

Based on successful structural studies, the following protocol has proven effective for isolating and purifying TBEV for structural analysis :

• Virus cultivation: Grow virus in appropriate cell culture systems (mammalian or tick cells depending on research focus)

• Inactivation: For safety, inactivate virus with 0.2% formaldehyde while preserving structural integrity

• Initial purification:

  • Iterative centrifugation

  • Resuspension of viral pellet

  • Additional centrifugation to concentrate virus

• Density gradient separation:

  • Centrifuge concentrated virus on a sucrose density gradient

  • Identify target fractions containing TBEV by ELISA

  • Collect and re-centrifuge these fractions

• Final preparation:

  • Resuspend viral pellet in TNE/5 buffer

  • Aliquot and store at -70°C

  • Assess concentration using established methods

For subsequent structural analysis, cryo-EM sample preparation includes:

• Application to grids

• Vitrification

• Data collection at 300 kV with appropriate detector settings

• Image processing with software such as Warp and CryoSPARC

This procedure has allowed for 3.0 Å resolution structures of the TBEV E protein ectodomain, though it should be noted that the C-terminal stem and anchor regions are often not well-resolved using this method .

How can researchers effectively analyze the evolutionary constraints on the TBEV E protein C-terminal region?

To analyze evolutionary constraints on the TBEV E protein C-terminal region, researchers should implement a multi-faceted approach:

• Comprehensive sequence compilation: Gather complete genome sequences from all TBEV subtypes and lineages. The TBEVnext dataset, which includes 225 TBEV strains spanning all subtypes and lineages, provides an excellent starting point .

• Phylogenetic analysis: Construct phylogenetic trees using maximum likelihood or Bayesian methods to establish evolutionary relationships. The Nextstrain framework has proven useful for visualizing TBEV phylogeography .

• Selection pressure analysis: Calculate the ratio of non-synonymous to synonymous substitutions (dN/dS) specifically for the C-terminal region to identify sites under positive or negative selection.

• Structural constraint mapping: Map conserved regions onto the 3D structure to identify functionally important sites. For the C-terminal region that may not be well-resolved in cryo-EM structures, computational modeling with AlphaFold2 or similar tools can be employed .

• RNA structure analysis: Investigate potential RNA secondary structures in the coding region that might constrain sequence evolution independently of protein function .

• Comparative biology approach: Compare constraints across different flaviviruses to identify TBEV-specific patterns versus general flavivirus requirements.

• Ancestral sequence reconstruction: Infer ancestral sequences to track the evolution of specific features in the C-terminal region.

Using these methods in combination provides a robust framework for understanding the evolutionary constraints acting on the TBEV E protein C-terminal region, potentially revealing insights into host adaptation, pathogenicity, and immune evasion strategies.

What are the main contradictions or knowledge gaps in current research on TBEV E protein's C-terminal region?

Several significant knowledge gaps and contradictions exist in the current understanding of TBEV E protein's C-terminal region:

• Structural characterization challenges: While high-resolution structures (3.0 Å) of the TBEV E protein ectodomain are available, the stem and anchor regions at the C-terminal end are poorly resolved in cryo-EM analyses . This is particularly true for formaldehyde-inactivated virus, where disorder in the transmembrane region is likely caused by the inactivation treatment .

• Host-dependent structural differences: While glycosylation patterns differ significantly between viruses grown in mammalian versus tick cells , it remains unclear how these differences specifically affect the C-terminal region's structure and function.

• Functional significance of quasispecies variations: Although sequencing has identified nucleotide variations in the E protein gene , the functional significance of mutations specifically in the C-terminal region is not well established.

• Correlation between structure and pathogenicity: Despite known differences in pathogenicity among TBEV subtypes , the specific contribution of C-terminal region variations to these differences remains poorly understood.

• Immune recognition and evasion: While studies have characterized CD8+ T cell responses to TBEV , the specific role of the C-terminal region in immune recognition and potential immune evasion strategies is not fully elucidated.

Addressing these gaps will require integrated approaches combining structural biology, immunology, molecular virology, and evolutionary analysis.

What novel experimental approaches might advance our understanding of TBEV E protein's C-terminal function in viral pathogenesis?

Several innovative approaches could significantly advance our understanding of the C-terminal region's function:

• Advanced structural methods:

  • Hydrogen-deuterium exchange mass spectrometry to probe flexible regions

  • Single-particle cryo-electron tomography to visualize membrane-embedded portions

  • Solid-state NMR for membrane-associated domains

  • Nanodiscs or amphipols to stabilize transmembrane regions for structural studies

• Genetic engineering approaches:

  • CRISPR-based screens in host cells to identify interaction partners

  • Viral reverse genetics creating chimeric viruses with C-terminal exchanges between subtypes

  • Site-directed mutagenesis targeting conserved features in the C-terminal region

  • Reporter viruses to track membrane fusion events mediated by the C-terminal region

• Advanced imaging techniques:

  • Super-resolution microscopy to track E protein dynamics during viral entry

  • Correlative light and electron microscopy (CLEM) to connect molecular events with ultrastructural changes

  • Live-cell imaging with fluorescently tagged E protein variants

• Systems biology approaches:

  • Integrative multi-omics analyses combining proteomics, glycomics, and structural data

  • Machine learning to identify patterns in sequence-structure-function relationships

  • Network analysis of host-pathogen protein interactions focused on the C-terminal region

• Translational approaches:

  • Structure-based design of inhibitors targeting the C-terminal region

  • Development of vaccines eliciting responses against conserved epitopes in the C-terminal region

  • Engineered antibodies specifically recognizing conformational states of the C-terminal region

These approaches would complement existing research methodologies and potentially overcome current limitations in studying this challenging but functionally important region of the TBEV E protein.