Recombinant Human herpesvirus 6B Envelope glycoprotein H (gH)

What is the role of glycoprotein H (gH) in HHV-6B infection?

Glycoprotein H (gH) is a critical component of the HHV-6B virus envelope that functions as part of a tetrameric complex gH/gL/gQ1/gQ2. This complex serves as the viral ligand for the cellular receptor human CD134 (hCD134, also known as OX40), which is specifically expressed on activated T cells. The interaction between this tetrameric complex and hCD134 is essential for virus entry into susceptible cells and is a key determinant of HHV-6B cell tropism. Unlike HHV-6A which binds to CD46 expressed widely on human cells, HHV-6B specifically recognizes hCD134 through its unique gH/gL/gQ1/gQ2 complex.

The gH/gL portion of the tetramer forms a structural base for the attachment of gQ1 and gQ2, with gQ1 directly interacting with the hCD134 receptor. This interaction differentiates HHV-6B from other herpesviruses and determines its specific infection of activated T cells.

How does the recombinant HHV-6B gH complex differ structurally from native viral gH?

Recombinant HHV-6B gH is typically produced as a soluble form by removing the transmembrane domain that anchors it to the viral envelope. In experimental settings, researchers have successfully created soluble forms of the tetrameric complex (gH/gL/gQ1/gQ2) by replacing the transmembrane domain of gH with cleavable tags such as human IgG1 Fc and His tags (gHFcHis). This modification allows the tetramer to be secreted from expression cells while maintaining its ability to bind to the hCD134 receptor.

When expressed in mammalian cell systems, the soluble recombinant tetramer maintains a similar conformation to the native complex, appearing as a monodispersed protein in size-exclusion chromatography and electron microscopy analyses. Single-particle analysis by negative-staining electron microscopy has revealed that the tetramer has an elongated shape with a gH/gL part and additional density corresponding to gQ1/gQ2. This structure enables the complex to correctly present binding sites for both neutralizing antibodies and the receptor.

What expression systems are most effective for producing recombinant HHV-6B gH?

Mammalian cell expression systems are considered most effective for producing recombinant HHV-6B gH and the complete tetrameric complex. This approach is preferred because the complicated hetero-tetrameric complex (gH/gL/gQ1/gQ2) requires proper assembly during its transit through host cell organelles while undergoing appropriate glycosylation.

For successful expression, researchers have utilized the following methodology:

• Codon optimization of the four genes (gH, gL, gQ1, and gQ2) from HHV-6B HST strain

• Subcloning of the gH ectodomain (residues 16-667) into expression vectors such as pFuse-hIG1-Fc2

• Construction of dual-gene expression vectors (e.g., pCAGGS-gHFcHis/gL or pCAGGS-gQ1/gQ2)

• Integration of selection marker cassettes (neomycin or puromycin resistance)

• Transfection into mammalian cells for stable expression

This process has been shown to yield functional recombinant tetramer that maintains binding affinity to hCD134 and neutralizing antibodies.

What are the optimal purification protocols for recombinant HHV-6B gH tetrameric complex?

Purification of the recombinant HHV-6B gH tetrameric complex requires a multi-step approach to achieve high purity while maintaining the complex's structural integrity and functionality. Based on published research, the following purification protocol has been successfully employed:

• Initial capture of the secreted tetrameric complex from cell culture supernatant using affinity chromatography, typically targeting the His-tag or Fc-fusion tag attached to the recombinant gH

• Further purification by size-exclusion chromatography to separate the intact tetrameric complex from incomplete assemblies or aggregates

• Verification of complex integrity and homogeneity using analytical techniques such as SDS-PAGE and Western blotting

The purified tetramer appears as a single peak in size-exclusion column chromatography, consistent with a monodispersed pattern observed in electron microscopy analysis. This indicates proper folding and assembly of the complex. The purified tetramer maintains its competency to interact with neutralizing monoclonal antibodies and the hCD134 receptor, demonstrating functional integrity after purification.

How can researchers accurately measure binding affinity between recombinant HHV-6B gH complex and CD134 receptor?

Surface plasmon resonance (SPR) analysis has been demonstrated as an effective method for quantitatively measuring the binding affinity between the soluble HHV-6B tetramer and its receptor hCD134. The methodology involves:

• Immobilization of recombinant hCD134 with C-terminal human FcHis-tag (replacing the transmembrane domain) on a sensor chip

• Loading purified soluble tetramer as the analyte

• Collection of response curves and calculation of kinetic parameters

Using this approach, researchers have determined that the HHV-6B tetramer binds to hCD134 with high affinity (KD = 18 nM), although the interaction exhibits fast dissociation/association kinetics. The same method can be used to measure the affinity of neutralizing antibodies to the tetramer, such as anti-gQ1 monoclonal antibody (KD = 17 nM) and anti-gH monoclonal antibody (KD = 2.7 nM).

Competition assays between antibodies and the receptor can also reveal which components of the tetramer directly interact with hCD134. For example, it has been shown that anti-gQ1 antibody competes with hCD134 for tetramer binding while anti-gH antibody does not, indicating that gQ1 directly interacts with the receptor.

What structural analysis techniques provide the best insights into recombinant HHV-6B gH conformation?

• The elongated shape of the tetrameric complex

• The distinct gH/gL portion and the additional density corresponding to gQ1/gQ2

• The binding sites for antibodies and the receptor

In EM studies, researchers have observed that anti-gQ1 antibody binds to the tip of the extra density (corresponding to gQ1/gQ2), while anti-gH antibody binds to the putative gH/gL part of the complex. These observations help map functional domains within the tetramer.

For more detailed structural analysis, researchers suggest combining EM with X-ray crystallography and cryo-electron microscopy to reveal finer details of the HHV-6B tetramer's unique features and their significance in viral infection. These advanced structural methods would build upon the macroscopic insights gained from negative-staining EM and potentially identify key residues involved in receptor binding and neutralizing epitopes.

How can recombinant HHV-6B gH be used to develop neutralizing antibodies?

Recombinant HHV-6B gH, particularly as part of the tetrameric complex, serves as an excellent immunogen for developing neutralizing antibodies. Researchers have successfully generated neutralizing monoclonal antibodies against different components of the tetramer, including:

• Anti-gQ1 antibodies (e.g., Mab KH-1) that compete with hCD134 for binding to the tetramer

• Anti-gH antibodies (e.g., Mab OHV-3) that bind to the gH/gL portion without competing with receptor binding

These antibodies exhibit high affinity for the tetramer (KD values of 17 nM and 2.7 nM for anti-gQ1 and anti-gH, respectively) and demonstrate neutralizing activity against HHV-6B infection.

The purified recombinant tetramer provides researchers with a well-defined antigen for immunization strategies, allowing the production of antibodies targeting specific components of the viral entry machinery. Characterization of these antibodies through techniques such as surface plasmon resonance and competition assays helps elucidate their mechanisms of action and potential therapeutic applications.

What is the potential of recombinant HHV-6B gH tetramer as a vaccine candidate?

The recombinant HHV-6B gH tetrameric complex has shown promising potential as a vaccine candidate against HHV-6B infection. Studies in mice have demonstrated that immunization with the purified tetramer, when administered with appropriate adjuvants, can induce both humoral and cellular immunity against HHV-6B.

A key experimental approach involves:

• Administration of purified recombinant tetramer with aluminum hydrogel adjuvant and/or CpG oligodeoxynucleotide adjuvant

• Multiple immunizations to strengthen the immune response

• Assessment of both antibody production and cellular immune responses

The rationale for targeting the tetramer in vaccine development is compelling because it represents the viral ligand essential for entry into host cells. Antibodies that block this interaction can potentially neutralize viral infectivity. Additionally, the tetramer contains multiple viral proteins (gH, gL, gQ1, and gQ2), potentially inducing broad immune responses against different viral epitopes.

How do mutations in recombinant HHV-6B gH affect its interaction with receptors and neutralizing antibodies?

Studies with recombinant herpesvirus glycoproteins have demonstrated that specific mutations can significantly alter interactions with receptors and neutralizing antibodies. While the provided search results don't specifically address mutations in HHV-6B gH, research on related herpesvirus glycoproteins (like HHV-6A gB) provides a methodological framework.

For example, in HHV-6A, introduction of lysine or alanine substitutions at asparagine 347 of glycoprotein B (gB) resulted in viable virus that was no longer recognized by a neutralizing monoclonal antibody (MAb 87-y-13). This demonstrates how single amino acid changes can affect antibody recognition while maintaining viral viability.

A similar experimental approach could be applied to study HHV-6B gH:

• Identification of key residues in gH or other components of the tetramer through sequence analysis and structural modeling

• Site-directed mutagenesis to create specific amino acid substitutions

• Expression of mutant tetramers and assessment of receptor binding and antibody recognition

• If possible, construction of recombinant viruses expressing the mutant proteins to evaluate effects on viral entry and growth

Such studies would help identify critical functional residues in the tetramer and potential escape mechanisms from neutralizing antibodies, informing both vaccine design and antiviral therapy development.

How do the gH complexes of HHV-6A and HHV-6B differ in structure and receptor specificity?

Despite their close phylogenetic relationship, HHV-6A and HHV-6B demonstrate distinct receptor specificities mediated by their respective gH/gL-based complexes. Both viruses form tetrameric complexes (gH/gL/gQ1/gQ2), but they recognize different cellular receptors:

• HHV-6A tetramer binds to CD46, which is widely expressed on human cells

• HHV-6B tetramer recognizes human CD134 (hCD134/OX40), specifically expressed on activated T cells

This receptor specificity difference is primarily determined by variations in the gQ1 and gQ2 components of the tetramers. While both viruses also form a trimeric complex (gH/gL/gO), the tetrameric complex plays the critical role in receptor recognition and host cell entry.

The distinct receptor tropism explains the different cellular targets and potentially the distinct pathogenesis of these two viruses. The molecular basis for this specificity lies in the conformation of gQ1 in cooperation with gQ2, along with the gH/gL base. Responsible residues in both HHV-6B gQ1 and hCD134 have been identified, though the complete structural details of these interactions require further elucidation.

What methodological challenges exist in studying recombinant HHV-6B gH interactions, and how can they be addressed?

Several methodological challenges exist in studying recombinant HHV-6B gH interactions, particularly related to the complex nature of the tetrameric assembly. These challenges and potential solutions include:

• Complex assembly and proper glycosylation: The tetrameric complex requires coordinated expression of four different proteins with proper folding and glycosylation. This necessitates mammalian expression systems rather than bacterial or insect cell systems. Researchers have addressed this by developing stable mammalian cell lines expressing all four components with appropriate signal sequences and tags for purification.

• Dynamic nature of receptor interaction: The fast dissociation/association kinetics between the tetramer and hCD134 presents challenges for detailed interaction studies. Surface plasmon resonance with careful experimental design has proven effective for quantifying these interactions despite their dynamic nature.

• Structural complexity: The large size and flexibility of the tetrameric complex make high-resolution structural analysis challenging. A multi-method approach combining negative-staining EM with X-ray crystallography of subdomains and cryo-EM has been suggested as the most promising strategy.

• Functional verification: Confirming that recombinant constructs maintain native functionality requires careful design of binding assays and, ideally, virus neutralization studies. Using multiple neutralizing antibodies as controls can help verify proper conformation of recombinant complexes.

How does the HHV-6B gH tetrameric complex compare to analogous structures in other herpesviruses?

The HHV-6B gH tetrameric complex (gH/gL/gQ1/gQ2) represents a specialized adaptation within the herpesvirus family, where gH/gL-based complexes serve as key mediators of viral entry. Comparative analysis reveals both common features and unique aspects:

These comparative insights highlight how the HHV-6B tetramer represents both conserved evolutionary features and virus-specific adaptations that determine its unique tropism and pathogenesis. Further structural studies would enhance understanding of these relationships and potentially reveal common mechanisms that could be targeted for broad antiviral strategies.

What high-throughput screening approaches can identify inhibitors of recombinant HHV-6B gH-receptor interactions?

The availability of purified recombinant HHV-6B tetramer opens possibilities for developing high-throughput screening (HTS) approaches to identify inhibitors of gH-receptor interactions. Although not explicitly covered in the search results, the following methodologies could be implemented based on the tetramer's characterized properties:

• Fluorescence-based binding assays: Development of FRET (Förster Resonance Energy Transfer) or fluorescence polarization assays using labeled tetramer and receptor to measure binding inhibition by test compounds. The demonstrated high affinity between the tetramer and hCD134 (KD = 18 nM) provides a suitable dynamic range for such assays.

• Surface plasmon resonance competition assays: Adaptation of the SPR methods used to characterize tetramer-receptor interactions for screening compounds that disrupt this binding. This would enable real-time monitoring of binding inhibition and provide kinetic information about inhibitor action.

• Cell-based reporter assays: Development of cell lines expressing hCD134 and a reporter system activated upon tetramer binding, allowing screening for compounds that block this interaction in a cellular context.

The recombinant tetramer's demonstrated quality and stability in biochemical assays suggest it would serve as a reliable reagent for such screening efforts, potentially leading to the identification of novel antivirals targeting the HHV-6B entry mechanism.

How might advances in structural biology techniques improve our understanding of recombinant HHV-6B gH function?

Recent advances in structural biology, particularly in cryo-electron microscopy (cryo-EM) and integrative structural biology approaches, offer significant opportunities to enhance our understanding of the HHV-6B tetramer's function. Potential applications include:

The search results indicate that further structural analyses using X-ray crystallography and cryo-electron microscopy have been recommended to unveil the HHV-6B tetramer's unique features and significance in infection, suggesting that the field is moving in this direction.