Recombinant Saimiriine herpesvirus 2 Glycoprotein H (gH)

Basic Research Questions

• What is glycoprotein H (gH) and what role does it play in herpesvirus entry?

  Glycoprotein H (gH) is an essential component of the conserved core fusion machinery in all members of the Herpesviridae family. This machinery consists of glycoprotein B (gB) and the gH/gL complex. In herpes simplex virus (HSV) entry, the current model indicates that receptor-binding by glycoprotein D (gD) signals the gH/gL heterodimer to trigger a refolding event in gB that ultimately fuses the viral envelope with the host cell membrane . gH/gL functions as a critical regulator that activates gB-mediated fusion rather than acting as a fusion protein itself. The gH protein typically exists as a heterodimer with gL, and this complex is essential for proper processing, transport, and function of gH in the viral entry process .

• How are the domains of glycoprotein H organized and what are their functions?

  Structural studies of gH proteins from different herpesviruses have identified four conserved domains:

  • Domain I: Located at the N-terminus, this domain is primarily responsible for species-specific interactions with gL. It is essential for proper folding and transport of gH .

  • Domains II and III: These central, predominantly α-helical domains appear to form an interdependent functional unit. Studies suggest that these domains do not tolerate major substitutions between different herpesvirus gH proteins, indicating their crucial role in maintaining the specific architecture required for proper gH function .

  • Domain IV: The C-terminal domain is the most highly conserved in sequence and structure across herpesviruses. This domain includes a membrane-proximal region that may undergo structural changes during fusion initiation, possibly involving movement of a conserved basic flap to unmask an underlying hydrophobic region for membrane interaction .

• What expression systems are commonly used for recombinant herpesvirus gH production?

  For experimental studies of herpesvirus gH, several expression systems have been successfully employed:

  • Bacterial expression systems: GST fusion proteins containing portions of gH (N-terminal or C-terminal) can be expressed in Escherichia coli. For example, GST fusion proteins containing either the N-terminal or C-terminal parts of HSV-1 gH have been expressed in E. coli XL1 Blue MRF after transformation with specific plasmids (pGEX-H1FgHN or pGEX-H1FgHC) .

  • Mammalian cell expression: For functional studies, gH is commonly expressed in mammalian cells such as rabbit kidney (RK13) cells using expression plasmids and transfection reagents like X-tremeGENE HP .

  • Viral vector systems: Bacterial artificial chromosomes (BACs) have been used to generate recombinant herpesviruses expressing modified versions of gH for investigating functional interactions in the context of viral infection .

Intermediate Research Questions

• How do researchers study the functional interactions between gH and other viral glycoproteins?

  Several experimental approaches are used to investigate gH interactions with other viral glycoproteins:

  • Cell-cell fusion assays: These assays involve co-expressing gH/gL with other viral glycoproteins (such as gB and gD) in cultured cells to quantify fusion activity. This approach allows researchers to measure how changes in gH affect its ability to promote membrane fusion in conjunction with other glycoproteins .

  • Virus passaging and selection: Viruses with mutations in gB that cause small plaque phenotypes can be serially passaged to select for second-site mutations (often in gH) that restore plaque size. This approach allows researchers to identify functional interaction sites between gH and gB .

  • Chimeric protein construction: By creating chimeric proteins containing domains from gH proteins of different herpesviruses, researchers can identify which domains are functionally conserved and which are involved in species-specific interactions .

  • Complementation assays: Cell lines stably expressing chimeric or mutant gH proteins can be tested for their ability to support replication of gH-deleted viruses, providing information about functional conservation and domain-specific activities .

• What techniques are used to analyze gH processing and transport to the cell surface?

  To assess proper processing and transport of gH proteins (wild-type or chimeric):

  • Western blotting: Used to analyze protein expression levels and post-translational modifications that indicate proper processing.

  • Immunofluorescence assays (IFA): RK13 cells can be cotransfected with expression plasmids for native or chimeric gH along with gL or other viral proteins. After fixation with paraformaldehyde and optional permeabilization with Triton X-100, cells are incubated with gH-specific antibodies followed by fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488-conjugated goat anti-rabbit IgG). The presence and localization of gH can then be visualized using fluorescence microscopy .

  • Surface expression analysis: By omitting the permeabilization step in immunofluorescence protocols, researchers can specifically detect gH proteins that have been successfully transported to the cell surface .

• How do researchers generate and characterize antibodies against herpesvirus gH?

  Production and characterization of anti-gH antibodies typically follows these steps:

  • Antigen preparation: GST fusion proteins containing N-terminal or C-terminal parts of gH are expressed in E. coli, purified, and used for immunization.

  • Immunization: Rabbits or other animals are immunized with the purified gH fragments following standard immunization protocols.

  • Antibody collection and purification: Antisera are collected and can be used directly or further purified to isolate the IgG fraction.

  • Validation: The specificity of the antibodies is validated using techniques such as Western blotting, immunofluorescence, and immunoprecipitation with both wild-type and mutant gH proteins .

Advanced Research Questions

• What approaches have been successful in creating functional chimeric gH proteins?

  Research on chimeric gH proteins has revealed important principles about domain functionality:

  • Domain-based substitutions: Creating chimeras by swapping entire domains between gH proteins from different herpesviruses has shown that some domains are more tolerant of substitution than others. For example, chimeric gH consisting of domains I to III from pseudorabies virus (PrV) and domain IV from HSV-1 retained significant fusion activity, demonstrating the functional conservation of domain IV across alphaherpesviruses from different genera .

  • Domain I and gL compatibility: Domain I exhibits species-specific interactions with gL. When domain I is swapped between herpesvirus species, the matching gL must also be provided to maintain function. For instance, chimeric gH containing domain I of HSV-1 and domains II to IV of PrV exhibited limited fusion activity only in the presence of HSV-1 gL, not PrV gL .

  • Domains II and III interdependence: These domains appear to form a functional unit that does not tolerate major substitutions. Chimeras with separate substitutions for domains II or III between HSV-1 and PrV resulted in complete loss of function, indicating their highly interdependent nature .

  • Species-specific adaptations: The interfaces between domains may represent adaptations that allow optimal interactions with homologous fusion proteins (gB) and other accessory proteins like gD .

• What mechanisms underlie the functional interaction between gH and gB during membrane fusion?

  The interaction between gH and gB is critical for herpesvirus entry, though the precise mechanisms remain under investigation:

  • Multiple interaction sites: Research using mutant viruses has identified multiple sites on gH that functionally interact with gB. These include position H789 in domain IV and S830 in the cytoplasmic tail of gH .

  • Membrane-proximal region: Domain IV of gH contains a conserved basic flap that may undergo conformational changes during fusion initiation, potentially unmasking a hydrophobic region for membrane interaction that could distort the viral envelope and prime it for fusion .

  • Species-specific interactions: The interaction between gH and gB appears to be species-specific. When SaHV-1 gB (which shares 65% sequence identity with HSV-1 gB) was expressed with HSV-1 gD and gH/gL, it showed only 15% of wild-type fusion levels, suggesting suboptimal interaction between these heterologous proteins .

  • Cytoplasmic tail involvement: Mutations in the cytoplasmic tail of gH (such as S830N) can rescue fusion function in viruses with altered gB, suggesting that the C-terminal regions of these proteins may directly interact during the fusion process .

• How can selective pressure be used to identify functional interaction sites between viral glycoproteins?

  Selective pressure approaches have proven valuable for identifying functional interaction sites:

  • Creation of fusion-impaired viruses: Using bacterial artificial chromosomes, researchers have generated HSV-1 mutants with impaired fusion due to changes in gB. These include replacing HSV-1 gB with SaHV-1 gB or introducing specific mutations (like the gB3A mutant with three alanine substitutions in domain V) .

  • Serial passage: The mutant viruses are serially passaged to select for second-site mutations that restore normal plaque size and fusion function .

  • Shifting selective pressure: To prevent selection for mutations in the originally modified protein, researchers can passage viruses in cells already expressing the mutant protein. For example, the gB3A virus was passaged in cells expressing gB3A to shift selective pressure toward other viral proteins .

  • Sequence analysis and functional validation: After passaging, viruses showing improved growth properties are sequenced to identify second-site mutations. These mutations are then introduced into expression constructs to verify their effects on fusion activity in isolation from other potential changes .

• What is known about the structural changes in gH during herpesvirus fusion activation?

  Understanding the structural changes in gH during fusion activation remains an active area of research:

  • Domain IV conformational changes: Evidence suggests that structural changes within domain IV during fusion initiation may lead to movement of a conserved basic flap to unmask an underlying hydrophobic region. This region might then interact with membranes to distort the viral envelope and prime it for fusion .

  • Mutation-induced effects: Specific mutations in gH, such as H789Y in domain IV, may alter the positioning of a membrane-proximal flap in the gH ectodomain, affecting its interaction with gB during fusion .

  • Cytoplasmic tail interactions: Mutations in the cytoplasmic tail of gH (like S830N) can partially rescue function of specific gB variants, suggesting that these cytoplasmic regions may interact during the fusion process. Rather than causing global hyperfusogenicity, these mutations appear to enhance interactions with specific gB variants .

  • Species-specific structural adaptations: The interfaces between domains II and III differ structurally in characterized gH homologs from different herpesviruses, potentially representing adaptations that facilitate optimal interactions with homologous fusion proteins .