HSV-1 gD (84-175)
Description
Introduction to HSV-1 gD (84–175)
HSV-1 gD is a 369-amino-acid type I transmembrane glycoprotein essential for viral entry. The ectodomain (residues 1–316) contains three N-linked glycosylation sites and binds receptors such as nectin-1, HVEM, and heparan sulfate . The 84–175 region spans part of this ectodomain and is recognized as an immunodominant epitope critical for antibody-mediated neutralization and vaccine development .
Functional Roles in Viral Entry
The 84–175 region is central to HSV-1’s entry process:
-
Receptor Binding:
-
Fusion Activation:
Immunological Significance
This region is a primary target for host immunity and vaccine strategies:
-
Neutralizing Antibodies: MAbs targeting 84–175 block gD’s interaction with receptors or gH/gL, inhibiting fusion . For example:
-
Vaccine Potential: Subunit vaccines using gD (e.g., ΔgD-2 viruses) have shown efficacy in preclinical models, though human trials have been limited .
Research Applications and Data
The 84–175 region is utilized in:
Neutralization Assays
Product Specs
Q&A
What functional roles does the HSV-1 gD (84-175) region play in viral entry?
The 84-175 domain of gD contains key structural motifs required for receptor binding and activation of the membrane fusion cascade. Crystallographic studies show that residues 84-175 form part of the immunoglobulin-like V-type core, which undergoes conformational changes upon nectin-1 or HVEM binding . Methodologically, truncation mutants lacking this region fail to mediate viral entry in nectin-1-expressing cells, as shown by plaque reduction assays using UL16-deficient HSV-1 strains . Surface plasmon resonance (SPR) experiments using recombinant gD (84-175) further demonstrate direct binding to soluble nectin-1 with a dissociation constant () of 12.3 nM .
Table 1: Functional assays for gD (84-175)
What contradictory evidence exists regarding gD (84-175)’s role in glycoprotein complex assembly?
Two competing models describe gD’s interactions with gH/gL and gB during fusion:
-
Sequential activation model: gD (84-175) binds receptors first, triggering transient interactions with gH/gL and gB .
-
Preassembled complex model: Stable gD-gH/gL-gB complexes exist pre-fusion, with receptor binding inducing conformational changes .
Key contradictions arise from split-fluorescent protein assays:
-
Supporting sequential activation: gH/gL-gB interactions increase 2.5-fold post-receptor binding .
-
Supporting preassembly: Förster resonance energy transfer (FRET) detects constitutive gD-gB proximity (<10 nm) independent of receptors .
Resolution strategy:
-
Use in situ crosslinking in live cells with membrane-impermeable reagents (e.g., BS³) to stabilize transient interactions.
-
Compare complex stability in UL16-knockout virions (impaired gD packaging) versus wild-type HSV-1 .
How do hyperfusogenic gB mutants alter dependency on gD (84-175) for membrane fusion?
Hyperfusogenic gB variants (e.g., A855V) bypass the need for gD-receptor binding when PILRα is present, but remain gD-dependent in nectin-1-mediated entry . Quantitative fusion assays using dual-split reporter cells show:
-
PILRα + gB(A855V): Fusion efficiency = 82% without gD.
This divergence suggests that gD (84-175) stabilizes gH/gL conformations required for nectin-1 signaling but not PILRα. To test this, employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) on gD-gH/gL complexes bound to each receptor.
What methodologies resolve conflicting data on gD (84-175)’s antigenic shielding by gC?
Studies conflict on whether gC sterically blocks antibody access to gD (84-175):
-
Supporting shielding: HSV-1ΔgC shows 4-fold higher anti-gD MAb neutralization sensitivity .
-
Contradictory data: Cryo-EM structures show no direct gC-gD contact in virions .
Experimental approach:
-
Protease accessibility assay: Treat purified virions with subtilisin, then quantify gD (84-175) degradation via SDS-PAGE.
-
Stochastic optical reconstruction microscopy (STORM): Map spatial distribution of gC and gD on virion surfaces.
Why do recombinant gD (84-175) antigens exhibit variable immunoreactivity across studies?
Batch-to-batch variability arises from:
-
E. coli expression artifacts: Misfolded aggregates form due to lack of eukaryotic glycosylation. Mitigate via refolding buffers containing 2 M urea and 5 mM reduced glutathione .
-
Epitope masking: Residual SDS (0.1%) in commercial antigens (e.g., ViroGen #00184-V) blocks 30% of linear epitopes. Pre-treat antigens with 0.5% Triton X-100 to restore antibody binding .
How to differentiate between gD’s receptor-binding versus fusogenic signaling roles using the 84-175 fragment?
Stepwise mutagenesis protocol:
-
Introduce alanine substitutions at residues R118 and K120 (critical for nectin-1 binding) .
-
Test mutant gD (84-175) in:
-
Receptor binding: SPR against nectin-1-Fc.
-
Fusion signaling: Co-transfect with gB/gH/gL into CHO-K1 cells; quantify syncytia.
-
Outcome:
-
R118A/K120A mutants lose receptor binding () but retain 74% fusion activity , confirming separable functions.
Can the gD (84-175) region be engineered to enhance oncolytic HSV-1 tropism?
Proof-of-concept studies inserted GD2-binding scFv into gD (84-175), but chimeric viruses failed entry due to steric hindrance . Solutions include:
-
Linker optimization: Insert 15-aa glycine-serine spacers between scFv and gD.
-
Hybrid receptors: Co-express GD2 with nectin-1 to exploit residual native gD function.
How do post-translational modifications in gD (84-175) affect antibody neutralization?
Unanswered due to technical limitations in detecting in situ modifications. Proposed workflow:
-
Immunoprecipitate gD from HSV-1-infected cell lysates using anti-84-175 MAbs.
-
Analyze via liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electron-transfer dissociation (ETD).
-
Correlate phosphorylation/O-GlcNAcylation sites with neutralization resistance in in vitro microneutralization assays.
Product Science Overview
Herpes Simplex Virus-1 (HSV-1) is a prevalent human pathogen known for causing oral and genital herpes. The virus has a complex structure and lifecycle, which includes both lytic and latent phases. One of the critical components of HSV-1 is glycoprotein D (gD), which plays a pivotal role in the virus’s ability to enter host cells.
Glycoprotein D (gD) is an essential envelope protein of HSV-1. It is involved in the initial stages of viral infection by mediating the virus’s attachment and entry into host cells. The gD protein interacts with specific receptors on the surface of host cells, facilitating the fusion of the viral envelope with the host cell membrane.
The recombinant form of HSV-1 gD, specifically the amino acid region 84-175, is a truncated version of the full-length protein. This region is known to contain immunodominant epitopes, which are crucial for eliciting an immune response. The recombinant gD (84-175 a.a.) is often produced in E. coli and used in various research and diagnostic applications .
- Vaccine Development: The recombinant gD (84-175 a.a.) is a key component in the development of vaccines against HSV-1. Its immunodominant epitopes make it an ideal candidate for inducing protective immunity.
- Diagnostic Tools: This recombinant protein is used in serological assays to detect HSV-1 infections. It helps in identifying the presence of antibodies against HSV-1 in patient samples.
- Research: The recombinant gD (84-175 a.a.) is widely used in research to study the mechanisms of HSV-1 entry and immune evasion. It serves as a valuable tool for understanding the virus’s interaction with host cells and the host immune response.
The production of recombinant gD (84-175 a.a.) typically involves cloning the gene segment encoding this region into an expression vector, which is then introduced into E. coli cells. The bacteria are cultured, and the recombinant protein is expressed and purified using various chromatographic techniques. The purified protein is then used in downstream applications .
