TBEV gE

What is the structural organization of TBEV glycoprotein E?

TBEV glycoprotein E (496 residues) is the major component of the mature viral particle and consists of four domains. Domain I forms a central β-barrel structure. Domain II is elongated and contains the dimerization interface with a buried surface area of 14.9 nm², as well as the only glycosylation site (Asn154). This domain also houses the highly conserved fusion loop (residues 100-109) critical for viral-host membrane fusion. Domain III has an immunoglobulin-like fold. All four domains are visible in cryo-EM reconstructions, providing valuable insights into the structural arrangement of this protein .

How does glycosylation affect TBEV gE function in different host systems?

The glycosylation pattern of TBEV gE varies significantly between mammalian and tick hosts. In human neuronal cells, complex and hybrid glycans (containing galactose, GlcNAc, mannose, and fucose) predominate on the viral surface. In contrast, TBEV grown in tick cells mainly displays paucimannose and high-mannose structures with five and six mannoses . This differential glycosylation has functional consequences: N-glycosylation is critical for virus egress from mammalian cells but dispensable in tick cells. While not essential for viral entry in cell culture, glycosylation at Asn154 contributes significantly to neurovirulence in mice, suggesting a potential role in neural tissue tropism .

What techniques are most effective for isolating and purifying TBEV gE for structural studies?

For TBEV gE isolation, researchers typically employ recombinant protein expression systems or direct purification from viral preparations. When using viral sources, purification protocols often involve:

• Virus concentration through ultracentrifugation

• Protein separation via SDS-PAGE

• In-gel processing of purified E protein bands

• Release of N-glycans using PNGase F (3 mU) at 37°C for 3 hours

• Sonication to enhance oligosaccharide release from gel pieces

For comparative studies, control samples from uninfected cells undergo parallel processing including ethanol washing, sonication, and extraction with chloroform-methanol-water solution (8:4:1 ratio) . Recombinant protein approaches offer an alternative that avoids biosafety concerns associated with infectious virus .

What methods enable detailed characterization of TBEV gE glycan structures?

Comprehensive glycan analysis of TBEV gE requires a multi-method approach:

• Enzymatic release: PNGase F treatment (37°C for 3 hours) efficiently releases N-linked glycans from purified E protein

• Extraction protocol: Chloroform-methanol-water solution (8:4:1 ratio, ~300 μL) followed by 5 minutes of sonication

• Centrifugation: 5 minutes at 3,000 × g to separate fractions

• Sample preparation: Evaporation of organic solvents under vacuum and resuspension in 5 mM ammonium bicarbonate

• Analytical techniques: Mass spectrometry for glycan composition determination and site-specific glycopeptide analysis

This workflow has successfully distinguished the glycosylation profiles of TBEV derived from human neuronal versus tick cells, revealing host-specific modifications that may influence pathogenicity.

How does TBEV gE contribute to viral attachment and entry?

TBEV gE facilitates viral attachment and entry through multiple mechanisms:

• Interaction with attachment factors: While TBEV utilizes various cellular attachment factors, heparan sulfate (a glycosaminoglycan) is particularly important. The binding between heparan sulfate and TBEV occurs via the E protein, with cell culture adaptations often manifesting as mutations that increase the positive charge of the E protein .

• Carbohydrate-mediated interactions: Although the carbohydrate moiety of TBEV gE is dispensable for entry in cell culture systems, its absence reduces neuroinvasiveness in mouse models. This suggests potential interactions between glycosylated Asn154 and carbohydrate-binding proteins on neuronal cells, which may explain tissue-specific pathogenicity .

• Fusion activity: The hydrophobic fusion loop (residues 100-109) in domain II is essential for merging viral and host cell membranes during entry. This loop remains hidden from the aqueous environment by residing in the hinge region between domains I and III of the adjacent E protein in the dimer and is further shielded by the carbohydrate moiety of residue 154 .

What experimental systems best model TBEV gE functional differences between strains?

Recent research has established several effective experimental systems for comparing TBEV strains:

• Human alveolar epithelial cells (A549): This cell line effectively differentiates growth kinetics between TBEV strains. For example, the divergent Salland strain (≥2% difference at amino acid level from other TBEV-Eu members) demonstrated growth patterns comparable to the highly pathogenic Hypr strain, both growing faster than the mildly pathogenic Neudoerfl strain .

• Human neuroblastoma cells (SK-N-SH): This neuronal model shows even greater strain differentiation, with the Salland strain replicating faster and achieving higher infectious titers than both reference strains .

• Primary human monocyte-derived dendritic cells: All three strains (Salland, Hypr, Neudoerfl) infected these primary cells to similar extents and showed comparable interactions with the type I interferon system, suggesting conserved mechanisms in immune cell interactions despite sequence divergence .

These systems can be utilized to examine how specific variations in the gE sequence correlate with functional phenotypes across different TBEV strains.

How can researchers interpret TBEV gE antibody responses in serological studies?

Serological studies of TBEV gE antibodies require careful interpretation due to several complexities:

• Vaccination versus infection: TBEV E protein-specific IgG prevalence reaches 72.1% (95% CI 68.2–75.7%) in vaccinated individuals compared to 6% (95% CI 4.4–7.8%) in unvaccinated individuals, necessitating vaccination history documentation in study populations .

• Differential antibody persistence: Unvaccinated individuals who have experienced natural infection show a 30.3% seroreversion rate within one year, nearly ten times higher than the rate observed in vaccinated individuals (3.4%, with an annual decline rate of 8.0%). This rapid decline may lead to underestimation of previous infection rates in cross-sectional studies .

• Complementary testing approaches: Combining TBEV envelope (E) protein IgG ELISA with TBEV non-structural protein 1 (NS1) IgG ELISA provides more complete serological profiles. NS1-specific IgG antibodies are six times more common in vaccinated than unvaccinated individuals, offering a potential marker to distinguish vaccination from natural infection .

• Confirmation methods: Seroneutralization testing remains essential for confirming seroconversions in unvaccinated individuals, particularly when monitoring infection incidence .

What structural insights guide antibody-based research targeting TBEV gE?

Structural analysis of antibody-TBEV gE interactions has revealed important insights for immunological research:

• Domain-specific targeting: The structure of the complex between the protective antibody ch14D5 and the D3 domain of TBEV glycoprotein E demonstrates specific domain targeting that contributes to neutralization efficacy .

• Glycan shield considerations: The glycosylation at Asn154 creates a carbohydrate shield that may protect certain epitopes from antibody recognition while potentially creating unique glycan-dependent epitopes .

• Cross-reactivity potential: The immunoglobulin-like fold of domain III contains epitopes that may show cross-reactivity with antibodies against related flaviviruses, necessitating careful specificity testing in antibody development .

• Structural states: TBEV gE exists in different conformational states during the virus lifecycle, particularly during the fusion process, presenting opportunities for targeting conformation-specific epitopes .

What factors drive TBEV emergence and how might gE contribute to changing epidemiological patterns?

Research into TBEV emergence has identified multiple factors potentially related to gE characteristics:

• Viral adaptation: Sequence variations in gE may contribute to enhanced transmission efficiency or pathogenicity, as suggested by the comparative analysis of divergent strains like Salland .

• Host range expansion: gE-mediated interactions with new host species might facilitate geographic spread, particularly through interactions with novel tick vectors or reservoir hosts .

• Multifactorial drivers: A comprehensive expert elicitation study identified 59 possible drivers of TBEV emergence grouped into eight domains, demonstrating the complex interplay of factors beyond viral protein characteristics alone .

• Research prioritization: Understanding how gE variations contribute to these emergence factors requires targeted studies focusing on structure-function relationships across different environmental and host contexts .

What technical challenges persist in high-resolution structural analysis of native TBEV gE?

Despite significant progress, several challenges remain in structural characterization of TBEV gE:

• Glycan heterogeneity: The variable glycosylation of TBEV gE, particularly differences between tick-derived and mammalian cell-derived virus, complicates crystallization and structural determination .

• Conformational dynamics: TBEV gE undergoes significant conformational changes during the virus lifecycle, particularly during fusion, making it difficult to capture all functionally relevant states .

• Membrane context: As an integral membrane protein, full-length TBEV gE includes a transmembrane domain that poses challenges for structural determination using traditional crystallographic approaches .

• Strain variations: The emergence of divergent strains like Salland (≥2% difference at amino acid level) requires comparative structural analysis to understand functional implications of sequence variation .

• Integration of data: Combining crystallography, cryo-electron tomography, glycomics, and functional studies presents data integration challenges but offers the most comprehensive structural understanding .