HSV-1 gD

What cellular receptors does HSV-1 gD interact with, and how do they influence viral tropism?

HSV-1 gD determines which cells can be infected by binding to several distinct cellular receptors:

• Herpesvirus entry mediator (HVEM)

• Nectin-1 and nectin-2

• Modified heparan sulfate generated by specific 3-O-sulfotransferases

Research using viral entry assays with various gD mutants demonstrates that nectins serve as the principal entry receptors for human cell lines of neuronal and epithelial origin, whereas HVEM or nectins can mediate entry into T lymphocyte lines . This receptor distribution explains the tissue tropism of HSV-1, with T cells and fibroblasts primarily infected via HVEM, while neurons typically utilize nectin-based entry .

Methodological approach: To determine the primary receptor for a specific cell type, researchers can use receptor-selective gD mutants in entry assays, selectively block individual receptors with antibodies, or employ siRNA knockdown of specific receptors followed by infection challenges.

What domains of HSV-1 gD are essential for function, and how tolerant are they to mutations?

HSV-1 gD exhibits variable tolerance for mutations across different regions:

• The N-terminus can accommodate long insertions without losing function

• Residues adjacent to the gD Ig-like V-type core tolerate shorter insertions (up to 15 amino acids), but not those greater than 60 amino acids

• The membrane-proximal region (residues 261-305) contains a critical "pro-fusion domain" essential for infectivity and cell-cell fusion

Amino acid substitutions at positions 215, 222, and 223 specifically impair interactions with nectin-1 and nectin-2 while preserving or enhancing HVEM binding, highlighting the distinct binding interfaces for different receptors .

How does HSV-1 gD trigger the viral fusion machinery?

HSV-1 entry requires a coordinated sequence of interactions among four essential glycoproteins:

• Initial binding of gD to a cellular receptor (HVEM, nectin-1, etc.)

• Conformational changes in gD that expose or activate the pro-fusion domain

• Signal transmission to the gH/gL complex

• Activation of gB to execute membrane fusion

Recent studies using split-luciferase interaction assays reveal that:

• A proportion of gH/gL and gB associate in the endoplasmic reticulum and are transported to the plasma membrane together

• In contrast, gD traffics independently to the cell surface

• Once at the plasma membrane, gD, gH/gL, and gB form a complex

• When gD binds its receptor, conformational changes within this preformed complex efficiently transmit the activating signal through gH/gL to gB

This "conformational cascade" model explains how the energy from receptor binding is transmitted through the glycoprotein complex to drive the fusion process.

How do specific mutations in gD affect receptor selectivity and fusion function?

Double or triple amino acid substitutions at positions 215, 222, and 223 in gD cause:

• Marked reduction in binding to nectin-1

• Corresponding inability to function in cell fusion or viral entry via nectin-1 or nectin-2

• Enhanced or maintained functional interactions with HVEM and modified heparan sulfate

These findings demonstrate that different domains of gD, with some overlap, are critical for functional interactions with each class of entry receptor . This receptor selectivity has important implications for developing HSV strains with modified tropism, such as oncolytic viruses targeting specific tumor types or attenuated vaccine vectors.

What is the structure and function of the pro-fusion domain in HSV-1 gD?

The pro-fusion domain (PFD, residues 261-305) exhibits several distinctive features:

• High proline content with defined spacing patterns conserved across HSV strains

• Contains minimal PXXP motifs that could potentially bind proteins with SH-3 domains

• Highly conserved among HSV-1 and HSV-2 strains, with partial conservation in pseudorabies virus

Experimental evidence for the PFD's critical role includes:

• The gD1-260-CD8 chimera (lacking the PFD) fails in both virus entry and cell-cell fusion assays despite maintaining receptor binding

• The receptor-negative gDΔ6-259 (containing primarily the PFD) acts as a dominant-negative inhibitor of HSV infectivity in a dose-dependent manner

These findings suggest the PFD functions as a critical switch that, following receptor binding, undergoes conformational changes to interact with and activate the gH/gL complex, thereby triggering the fusion cascade.

How do HSV-1 glycoproteins interact before and during membrane fusion?

Advanced split-luciferase (NanoBiT) interaction assays in live cells have revealed critical insights into the temporal dynamics of glycoprotein interactions:

• gH/gL and gB interact at a steady level before and during fusion

• This interaction is detected even in the absence of target cells

• No significant change in gH/gL-gB interaction is observed upon addition of nectin-1-expressing target cells that trigger fusion

To investigate the domains involved in these interactions, researchers created constructs with:

• Scrambled HSV-1 gH cytoplasmic tail to disrupt interactions with the gB cytoplasmic domain

• Chimeric proteins combining domains from different herpesviruses

• Multiple tag combinations to optimize detection sensitivity

These experiments demonstrate that while glycoprotein complex formation is necessary for fusion, it is not sufficient, as all domains of gH/gL proved essential for fusion activity. Additionally, the data indicate that gH and gB interact in the endoplasmic reticulum, whereas gH and gD do not associate until reaching the plasma membrane .

What techniques are most effective for studying HSV-1 gD structure-function relationships?

Several complementary approaches have proven valuable for investigating gD structure and function:

• Mutagenesis strategies:

  • Site-directed mutagenesis targeting specific residues (positions 215, 222, 223 proved critical for nectin binding)

  • Domain deletion constructs (e.g., gD1-260 to remove the pro-fusion domain)

  • Domain swapping and chimeric proteins (e.g., gD1-260-CD8 fusion)

• Protein-protein interaction assays:

  • Split-luciferase complementation assays for real-time monitoring of interactions

  • Co-immunoprecipitation to detect stable complexes

  • Surface plasmon resonance for binding kinetics

• Functional assessment:

  • Cell-cell fusion assays using cells co-transfected with gD, gB, gH, and gL

  • Complementation of gD-negative virus with wild-type or mutant gD

  • Dominant-negative inhibition studies with non-functional gD constructs

The most informative approach combines structural analysis with functional validation to establish causative relationships between specific molecular features and biological activities.

How can researchers develop inhibitors targeting HSV-1 gD-receptor interactions?

Structure-based drug design targeting gD represents a promising antiviral strategy:

• Computational approaches:

  • Protein-protein docking and dynamic simulations of gD-HVEM and gD-Nectin-1 complexes

  • Identification of stable complexes and pivotal residues for receptor anchoring

  • Structure-based virtual screening against the gD binding interface

• Chemical library design:

  • Focus on scaffolds with demonstrated binding potential, such as triazolo[4,5-b]pyridines

  • Evaluate structure-activity relationships (SARs)

  • Optimize lead compounds for binding affinity and specificity

• Validation methods:

  • Binding assays using recombinant proteins

  • Cell-based entry inhibition assays

  • Assessment of broad-spectrum activity against different HSV strains

Research has identified several triazolo[4,5-b]pyridines with good theoretical affinity toward multiple conformations of HSV-1 gD, suggesting promising leads for developing antivirals that prevent viral attachment and penetration into host cells .

What systems can be used to study the dynamics of HSV-1 glycoprotein complexes?

Investigating the temporal and spatial dynamics of glycoprotein interactions requires sophisticated experimental systems:

• Live-cell imaging platforms:

  • Split-reporter protein complementation assays (e.g., NanoBiT) to monitor protein-protein interactions in real-time

  • Fluorescence resonance energy transfer (FRET) for detecting interactions within 10nm

  • Single-molecule tracking of fluorescently labeled glycoproteins

• Inducible fusion systems:

  • Co-culture assays with receptor-expressing target cells added to trigger fusion

  • Temperature-sensitive mutants that allow synchronization of the fusion process

  • Chemical induction of fusion by modulating protein conformation

• Domain mapping strategies:

  • Disruption of specific domains (e.g., scrambling the HSV-1 gH cytoplasmic tail)

  • Testing interactions between glycoproteins from different herpesviruses (e.g., EBV gH/gL with HSV-1 gB)

  • Systematic truncation of protein domains to identify interaction regions

By combining these approaches, researchers have established that HSV-1 glycoprotein complexes form before fusion, interact at a steady level throughout the fusion process, and do not depend on the presence of the fusion trigger for initial complex formation .

How might HSV-1 gD mutants with altered receptor specificity be used in vaccine development?

HSV strains carrying gD mutations that prevent entry via nectins may have significant potential as vaccine candidates because:

• They can establish transient infections in humans via HVEM-expressing cells (T cells and fibroblasts)

• They would likely be unable to establish latent infections in neurons, which primarily use nectin-1 for entry

• This attenuated phenotype provides a safety profile suitable for live virus vaccines

Research priorities for developing such vaccines include:

• Comprehensive characterization of receptor usage in different human tissues

• Assessment of immunogenicity and protective efficacy in animal models

• Determination of the optimal balance between attenuation and immunogenicity

• Evaluation of potential reversion to wild-type receptor usage

What are the prospects for developing targeted oncolytic HSV vectors based on gD modifications?

The plasticity of gD to tolerate insertions offers opportunities for engineering oncolytic HSV vectors:

• The N-terminus of gD can accommodate large insertions, potentially including targeting ligands

• Recombinant HSV-1 containing antibody fragments (e.g., ch14.18 scFv) at the N-terminus of gD has been attempted, though additional optimization is needed

• Pairing modified gD with hyperfusogenic gB mutants may enhance the spread and cytotoxic effects of oncolytic viruses

Future work should focus on:

• Optimizing the size and positioning of targeting ligands within gD

• Combining receptor retargeting with enhanced fusion capabilities

• Developing systems to evaluate tumor-specific entry and spread

• Assessing safety profiles through selective tropism studies

How can structural knowledge of the gD pro-fusion domain inform new antiviral strategies?

The pro-fusion domain (PFD) represents a promising target for antiviral development:

• It contains conserved elements across HSV-1, HSV-2, and related viruses

• Its distinctive features include high proline content and conserved spacing patterns

• The PFD functions as a critical switch in activating the fusion machinery

Novel therapeutic approaches might include:

• Designing peptide inhibitors that mimic interaction partners of the PFD

• Developing small molecules that bind to the PFD and prevent conformational changes

• Creating antibodies or antibody fragments that specifically recognize and block this region

• Identifying the key interaction partners of the PFD as additional drug targets

Understanding the structural transitions in this domain during fusion activation will be crucial for developing effective inhibitors that block the conformational cascade leading to viral entry.