Recombinant Feline herpesvirus 1 Envelope glycoprotein H (gH)

What is the role of glycoprotein H in FeHV-1 viral entry and pathogenesis?

Glycoprotein H (gH) is a crucial envelope protein that functions primarily in viral fusion and entry into host cells. In FeHV-1, as with other alphaherpesviruses, gH typically forms a heterodimeric complex with glycoprotein L (gL), creating the gH/gL complex that works in concert with glycoprotein B (gB) to mediate membrane fusion. This process is essential for viral infection of host cells. The gH protein contains domains that undergo conformational changes during the fusion process, facilitating the merger of viral and cellular membranes. Similar to the mechanisms observed in other herpesviruses, FeHV-1 gH likely plays a significant role in tissue tropism and virulence, affecting both the initial infection and viral spread within the feline host .

What expression systems are most effective for producing recombinant FeHV-1 gH?

For recombinant production of FeHV-1 gH, several expression systems have proven effective, each with specific advantages depending on research objectives. Mammalian cell systems, particularly Crandell-Rees feline kidney (CRFK) cells, are frequently used due to their ability to perform proper post-translational modifications essential for glycoprotein function. These modifications include correct folding, glycosylation patterns, and disulfide bond formation that might not occur properly in bacterial systems. For high-yield production, baculovirus expression systems in insect cells represent another viable option, offering a balance between proper protein modification and production scale. When selecting an expression system, researchers should consider whether the recombinant gH will be used for structural studies, immunological investigations, or as part of a recombinant vaccine construct, as these applications may require different levels of protein authenticity and yield .

What are the optimal methods for generating recombinant FeHV-1 expressing modified gH?

Generating recombinant FeHV-1 expressing modified gH typically employs homologous recombination techniques. The methodology involves several crucial steps: First, design an expression cassette containing the modified gH gene under control of a strong promoter, such as the human cytomegalovirus (HCMV) immediate-early promoter, which has proven effective in other recombinant FeHV-1 constructs. Second, flank this cassette with FeHV-1 sequences homologous to the desired insertion site, often the thymidine kinase (TK) gene, which allows for selection without compromising essential viral functions. Third, co-transfect this construct with wild-type FeHV-1 DNA into permissive cells (typically CRFK cells). Fourth, select recombinant viruses using either marker genes or, if the TK gene is disrupted, selection with nucleoside analogs like bromodeoxyuridine. Finally, plaque-purify the recombinant viruses and verify correct insertion through PCR, sequencing, and protein expression analysis. This approach has been successfully used with other FeHV-1 glycoproteins and FeLV genes, providing a template for gH modification .

How can researchers verify proper expression and localization of recombinant gH in FeHV-1?

Verification of proper expression and localization of recombinant gH requires a multi-faceted approach. Western blotting using antibodies specific to gH or to epitope tags engineered into the recombinant protein provides initial confirmation of expression and approximate molecular weight. To determine glycosylation status, which is crucial for proper gH function, researchers should employ endoglycosidase treatments such as Endo H and PNGase F, followed by Western blot analysis to observe mobility shifts. This approach helps distinguish between immature ER-associated forms and mature, post-Golgi forms of the glycoprotein, as demonstrated with other herpesvirus glycoproteins. Immunofluorescence assays are essential for confirming proper cellular localization, which should include membrane surface expression for functional gH. Flow cytometry can quantify surface expression levels, while transmission electron microscopy with immunogold labeling can verify incorporation of gH into viral particles. Functional validation should include cell-cell fusion assays, as gH is crucial for this process, and viral entry assays to confirm that the recombinant gH supports infection .

What techniques are most effective for studying gH interactions with other viral glycoproteins?

For studying gH interactions with other viral glycoproteins, particularly gL and gB, several complementary techniques yield comprehensive data. Co-immunoprecipitation assays remain the gold standard for detecting protein-protein interactions in near-native conditions. These assays can identify whether recombinant gH properly forms the essential heterodimer with gL and interacts with gB during the fusion process. Bimolecular fluorescence complementation (BiFC) provides spatial information about these interactions within living cells, revealing where in the cell these complexes form. Surface plasmon resonance (SPR) or bio-layer interferometry can determine binding kinetics and affinity constants between purified gH and its partners. For structural insights, cryo-electron microscopy has revolutionized the visualization of glycoprotein complexes, though this requires significant technical expertise. Functional fusion assays using cells expressing various combinations of glycoproteins help establish which interactions are essential for membrane fusion. Finally, proximity ligation assays can detect interactions in fixed cells with high sensitivity, offering advantages when protein expression levels are low .

How do specific mutations in the central helical domain of gH affect its fusion function in FeHV-1?

Mutations in the central helical domain of glycoprotein H can profoundly impact its fusion functionality, as demonstrated in related alphaherpesviruses. When making structural predictions for FeHV-1 gH mutations, the experience with other herpesvirus glycoproteins is instructive. For instance, proline substitutions in central helical regions of glycoproteins like gB cause significant structural distortions that impair protein trafficking and function. In the case of FeHV-1 gH, mutations disrupting the alpha-helical structure would likely prevent proper folding, leading to retention in the endoplasmic reticulum and failure to form functional complexes with gL. This would manifest as reduced surface expression, impaired cell-cell fusion activity, and ultimately diminished viral infectivity. Conservative substitutions that preserve helical structure might maintain basic functionality but could alter fusion kinetics or thermostability of the protein. Researchers investigating such mutations should employ a combination of predictive structural modeling (such as AlphaFold2), biochemical trafficking assays, and functional fusion assays to comprehensively characterize the effects of specific mutations .

What is the impact of glycosylation patterns on FeHV-1 gH immunogenicity and function?

Glycosylation patterns significantly influence both the immunogenicity and functionality of FeHV-1 glycoprotein H. N-linked glycans on herpesvirus envelope glycoproteins serve multiple crucial functions: they ensure proper protein folding, modulate protein-protein interactions essential for fusion complexes, and can shield immunodominant epitopes from neutralizing antibodies. For FeHV-1 gH, alterations in glycosylation sites through site-directed mutagenesis typically result in one of three outcomes: complete loss of expression due to protein misfolding, reduced trafficking to the cell surface despite protein expression, or altered fusion kinetics despite normal trafficking. Experimentally, comparing wild-type gH with glycosylation site mutants reveals that certain glycans may be dispensable for function while others are essential. From an immunological perspective, deglycosylated or differently glycosylated forms of gH often expose epitopes that are normally hidden, potentially generating more potent neutralizing antibody responses. This phenomenon could be exploited in recombinant vaccine design, where strategic modification of glycosylation sites might enhance protective immunity against FeHV-1 .

How can researchers reconcile contradictory data on gH neutralizing epitopes across different FeHV-1 strains?

When faced with contradictory data regarding neutralizing epitopes on gH across different FeHV-1 strains, researchers should implement a systematic analytical approach. First, sequence all gH genes from the strains in question to identify specific polymorphisms that correlate with differences in neutralization sensitivity. Second, generate a panel of monoclonal antibodies against recombinant gH and map their epitopes through techniques such as peptide scanning, competition assays, and neutralization escape mutant analysis. Third, create chimeric gH proteins by swapping domains between neutralization-sensitive and resistant strains to pinpoint the specific regions responsible for the observed differences. Fourth, utilize structural modeling to predict how identified polymorphisms might alter surface epitope presentation. Fifth, perform neutralization assays using a standardized protocol across multiple laboratories to eliminate methodological variations as a source of discrepancy. Finally, consider viral factors beyond gH sequence, such as differences in glycosylation machinery or the influence of other viral proteins on gH conformation. This comprehensive approach can identify whether contradictions stem from true biological differences between strains or from methodological inconsistencies .

How should researchers interpret apparent differences in gH trafficking between in vitro expression systems and natural infection?

When interpreting differences in gH trafficking between in vitro expression systems and natural infection, researchers must consider multiple factors that could account for these discrepancies. First, evaluate whether the expression level of gH differs significantly between systems, as overexpression can saturate cellular trafficking machinery and lead to artificial retention in the endoplasmic reticulum. Second, analyze the co-expression of other viral proteins, particularly gL, which forms an essential complex with gH and facilitates its proper folding and transport. The absence of gL in some expression systems may explain trafficking defects not seen during natural infection. Third, consider cell type-specific differences in glycosylation patterns and chaperone protein availability that might affect gH maturation differently in laboratory cell lines versus the natural host cells. Fourth, examine the influence of viral proteins other than gL that might modify trafficking pathways during infection but are absent in isolated expression systems. Finally, assess whether differences in protein detection methods across studies (such as antibody specificity or sensitivity) could create apparent rather than actual differences in trafficking patterns. A comparative study directly examining gH trafficking in parallel under different conditions would be the most definitive approach to resolving such discrepancies .

How can recombinant FeHV-1 expressing modified gH be utilized in vaccine development?

Recombinant FeHV-1 expressing modified gH presents several promising avenues for vaccine development. The approach can follow a rational design strategy where specific modifications to gH enhance immunogenicity while potentially attenuating the virus. For instance, researchers can engineer gH mutations that preserve immunodominant neutralizing epitopes but reduce fusion efficiency, creating a self-limiting virus that induces robust immunity. Based on successful approaches with other glycoproteins, inserting additional neutralizing epitopes from variant strains into non-essential regions of gH could generate broader cross-protection against multiple FeHV-1 strains. The recombinant virus approach offers significant advantages over subunit vaccines, as it presents gH in its native conformation and context, including proper associations with gL and other viral glycoproteins. This strategy stimulates both humoral and cell-mediated immunity, which is crucial for protection against herpesviruses. Examples from recent calicivirus work demonstrate that recombinant FeHV-1 vaccines expressing foreign antigens can induce robust immune responses while significantly reducing clinical disease scores and viral shedding following challenge .

What are the most promising approaches for studying the role of gH in FeHV-1 latency and reactivation?

Studying the role of gH in FeHV-1 latency and reactivation requires innovative approaches targeting both in vitro and in vivo systems. Developing a reproducible in vitro latency model using neuronal cultures derived from feline trigeminal ganglia provides a controlled environment to monitor gH expression during latency establishment, maintenance, and reactivation. Complementing this with targeted RNA interference methods, similar to those successfully employed against glycoprotein D, allows temporal control over gH expression at different stages of the viral lifecycle. Chromatin immunoprecipitation sequencing (ChIP-seq) techniques can identify epigenetic modifications regulating gH transcription during latency versus lytic infection. For in vivo studies, establishing a feline model with recombinant FeHV-1 expressing reporter genes linked to the gH promoter enables non-invasive tracking of promoter activity during latency and reactivation. Additionally, creating recombinant viruses with conditional expression of gH can determine precisely when this glycoprotein is required during reactivation from latency. This comprehensive approach would significantly advance understanding of how gH contributes to the complex latency-reactivation cycle that characterizes alphaherpesvirus pathogenesis .

How might CRISPR/Cas9 genome editing advance the study of gH function in FeHV-1?

CRISPR/Cas9 genome editing represents a revolutionary approach for studying gH function in FeHV-1, offering unprecedented precision in genetic manipulation. This technology enables several advanced experimental strategies: First, introducing specific point mutations to create a library of FeHV-1 variants with altered gH proteins, allowing systematic assessment of structure-function relationships. Second, inserting fluorescent tags at the endogenous gH locus to track the protein under truly native expression conditions without overexpression artifacts. Third, creating conditional knockouts where gH expression can be temporally controlled, revealing its role at different stages of infection. Fourth, engineering reporter constructs into the viral genome that activate specifically when gH interacts with partner proteins, providing real-time visualization of these interactions. Fifth, modifying glycosylation sites with single-nucleotide precision to assess their individual contributions to gH function. The methodology requires designing guide RNAs targeting specific regions of the gH gene, creating repair templates carrying the desired modifications, and delivering these components alongside Cas9 into cells infected with FeHV-1. Selection markers flanked by recombinase recognition sites allow for marker-free gene editing after selection. This approach dramatically accelerates the pace of discovery compared to traditional homologous recombination methods .

How do findings on FeHV-1 gH compare with studies on glycoprotein D in viral entry and immunogenicity?

Comparative analysis of FeHV-1 glycoproteins H and D reveals both complementary and distinct roles in viral pathogenesis and immunity. Glycoprotein D functions primarily as a receptor-binding protein and is a potent inducer of virus-neutralizing antibodies, making it a primary target for vaccine development. Research has demonstrated that glycoprotein D is necessary for viral infection, as evidenced by RNA interference studies that showed 77-85% reductions in viral mRNA and 84-77% reductions in viral titers when glycoprotein D expression was inhibited. In contrast, gH typically does not directly bind cellular receptors but instead works in conjunction with gL and gB to execute membrane fusion after receptor binding has occurred. While both glycoproteins are targets for neutralizing antibodies, the neutralizing epitopes on gH tend to be more conformationally dependent and may be partially shielded in the prefusion state. From an immunological perspective, vaccines targeting glycoprotein D often generate strong antibody responses but may offer incomplete protection against infection or reactivation, suggesting that a combinatorial approach including both gD and gH might yield more comprehensive immunity .

What parallels can be drawn between FeHV-1 gH research and studies on other veterinary herpesvirus glycoproteins?

Research on FeHV-1 glycoprotein H shares significant parallels with studies on glycoproteins from other veterinary herpesviruses, particularly those affecting domestic animals. Across bovine herpesvirus-1 (BoHV-1), equine herpesvirus-1 (EHV-1), and FeHV-1, the gH-gL complex serves as a conserved component of the fusion machinery, though with species-specific variations that determine host range. In vaccine development, recombinant approaches using the viral backbone to express modified glycoproteins have shown promise across these veterinary herpesviruses. For example, the successful development of recombinant FeHV-1 expressing calicivirus antigens mirrors similar strategies with BoHV-1 and EHV-1 vectors. A common challenge across these systems is balancing attenuation for safety while maintaining immunogenicity. Another parallel lies in the approaches to studying latency, where the establishment of latent infection in sensory neurons appears mechanistically similar across these viruses, though with species-specific triggers for reactivation. These similarities allow researchers to adapt successful methodologies across different veterinary herpesvirus systems, accelerating progress in understanding fundamental aspects of alphaherpesvirus biology .

What insights from human herpesvirus gH research might apply to FeHV-1 studies?

Research on human herpesvirus glycoprotein H offers valuable insights potentially applicable to FeHV-1 studies. Structural studies of HSV and VZV gH-gL complexes have revealed domain organizations and conformational changes during fusion that likely parallel those in FeHV-1 gH, providing templates for modeling and rational design of experiments. The identification of critical helical regions in human herpesvirus glycoproteins, particularly the finding that proline substitutions in central helices disrupt protein trafficking and function, directly informs similar studies in FeHV-1. For instance, research demonstrating that the V528 position in VZV gB (analogous to positions in the central helix of other glycoproteins including gH) is crucial for protein biogenesis suggests conservation of this feature across the Herpesviridae family. Studies on human herpesvirus glycoprotein epitopes recognized by neutralizing antibodies provide a roadmap for similar epitope mapping in FeHV-1 gH. Additionally, human herpesvirus research has pioneered techniques for studying glycoprotein interactions during fusion that can be adapted for FeHV-1, such as using split fluorescent proteins to visualize complex formation or developing cell-based fusion assays specific to feline cells .

What novel methodologies can improve the efficiency of generating recombinant FeHV-1 with modified gH?

Recent methodological advancements offer significant improvements for generating recombinant FeHV-1 with modified gH. The traditional homologous recombination approach, while effective, can be enhanced through several innovations. First, implementing CRISPR/Cas9-assisted recombination dramatically increases insertion efficiency by creating targeted double-strand breaks at the desired integration site. Second, developing bacterial artificial chromosome (BAC) systems for FeHV-1, similar to those used for other herpesviruses, enables precise genetic manipulation in bacterial systems before virus reconstitution in mammalian cells. Third, employing galK positive/negative selection in BAC systems allows marker-free modifications, eliminating concerns about selection markers affecting viral behavior. Fourth, utilizing Gibson Assembly or similar seamless cloning techniques enables the construction of complex expression cassettes with multiple modifications in a single step. Fifth, implementing inducible promoter systems to control gH expression provides a safeguard when testing potentially lethal mutations. These methodological improvements collectively reduce the time from design to characterization of recombinant viruses from months to weeks, significantly accelerating research progress in understanding gH function and developing potential vaccine candidates .

How can single-molecule techniques advance understanding of gH conformational changes during fusion?

Single-molecule techniques offer unprecedented insights into the dynamic conformational changes of gH during the fusion process. Förster resonance energy transfer (FRET) microscopy with strategically placed fluorophores on recombinant gH can detect nanometer-scale movements between domains during fusion activation, revealing the sequence and kinetics of conformational changes previously invisible to ensemble methods. Single-molecule force spectroscopy using atomic force microscopy can directly measure the energy landscape of gH as it transitions between prefusion and postfusion states, identifying energy barriers that could be targeted by antiviral drugs. By combining these approaches with total internal reflection fluorescence (TIRF) microscopy, researchers can visualize individual gH molecules in real-time as they respond to gD binding and interact with gB on the virion surface. Hydrogen-deuterium exchange mass spectrometry provides complementary structural information by identifying regions of gH that become exposed or protected during fusion activation. These single-molecule approaches overcome the limitations of crystallography and cryo-EM, which capture static snapshots, by revealing the dynamic process of how gH mediates membrane fusion in the context of the full fusion machinery .