Recombinant Saimiriine herpesvirus 2 Envelope glycoprotein B (gB)

What is the structural organization of SaHV-2 glycoprotein B and how does it compare to other primate herpesvirus gB proteins?

Glycoprotein B (gB) of Saimiriine herpesvirus 2, like other alphaherpesvirus gB proteins, functions as a class III membrane fusion protein that combines characteristics of both class I and II fusion proteins similar to vesicular stomatitis virus (VSV) fusion glycoprotein G . The protein structure is predicted to form a stable trimer in its pre-fusion state and undergoes significant conformational changes during the fusion process after interactions with other glycoproteins and cellular receptors .

While specific SaHV-2 gB crystal structures have not been fully characterized, comparative analysis with HSV-1 gB (one of the most conserved glycoproteins across herpesviruses) suggests a high degree of structural conservation in the core fusion machinery domains . Based on the homology observed between HSV-1 and SaHV-1 gB proteins, we can reasonably predict that SaHV-2 gB maintains similar structural features, including a long ectodomain with multiple glycosylation sites and a transmembrane region that anchors the protein in the viral envelope .

How does SaHV-2 gB contribute to the viral fusion mechanism and what other viral proteins are required?

SaHV-2 gB, similar to HSV-1 gB, likely serves as the principal membrane fusogen in what is termed the viral "fusion machine" complex . The fusion mechanism is highly regulated and requires multiple glycoproteins working in concert. For effective membrane fusion, gB must interact with glycoproteins gD, gH, and gL to form the minimum fusion complex . While not directly stated for SaHV-2, evidence from related viruses suggests that additional membrane glycoproteins, particularly glycoprotein K (gK), play critical roles in modifying gB-mediated membrane fusion .

The fusion process follows a coordinated sequence: gD binds to cellular receptors, triggering conformational changes that activate the gH/gL heterodimer, which in turn activates gB to execute the actual membrane fusion . During this process, gB transitions from its pre-fusion conformation to an extended intermediate state and finally to a post-fusion form, bringing the viral envelope and target cell membrane into proximity for fusion .

What expression systems are most appropriate for producing recombinant SaHV-2 gB with proper folding and post-translational modifications?

The expression of correctly folded and functionally active recombinant SaHV-2 gB requires careful consideration of post-translational modifications, particularly glycosylation. Based on knowledge of other herpesvirus gB proteins, mammalian expression systems are generally preferred over bacterial or insect cell systems to ensure proper glycosylation patterns. Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells have proven effective for expressing other primate herpesvirus glycoproteins with native-like properties .

When designing expression constructs, researchers should consider:

• Including the native signal sequence or a mammalian secretion signal for proper trafficking

• Preserving all potential N-linked and O-linked glycosylation sites

• Potentially truncating the transmembrane domain and cytoplasmic tail for improved secretion if soluble forms are desired

• Incorporating purification tags (His, FLAG, etc.) that minimally interfere with protein folding

The glycan moieties of viral glycoproteins play essential roles in viral binding, entry, transmission, and evasion of the host immune system, making proper glycosylation critical for functional studies .

What are the critical considerations when designing purification protocols for recombinant SaHV-2 gB?

Purifying recombinant SaHV-2 gB while preserving its native conformation requires careful protocol design. Based on experiences with related herpesvirus glycoproteins, a multi-step purification approach is recommended:

• Initial capture: Affinity chromatography using nickel columns for His-tagged constructs or anti-FLAG for FLAG-tagged proteins

• Intermediate purification: Ion-exchange chromatography to separate based on charge differences

• Polishing: Size-exclusion chromatography to isolate properly folded trimeric forms from aggregates or monomers

Special considerations include:

• Maintaining physiological pH (7.2-7.4) throughout purification

• Including stabilizing agents such as low concentrations of non-ionic detergents for full-length constructs

• Avoiding harsh elution conditions that might disrupt protein conformation

• Validating the trimeric state using analytical ultracentrifugation or native PAGE

The purification strategy should be tailored to the specific research questions being addressed, with more stringent purity requirements for structural studies compared to functional assays.

What cell-based assays are available to evaluate the fusion activity of recombinant SaHV-2 gB?

Several complementary assays can be employed to assess the fusion activity of recombinant SaHV-2 gB:

• Cell-cell fusion assays: Co-expressing SaHV-2 gB with gD, gH, and gL in one cell population and the appropriate receptors in another cell population. Fusion can be quantified using reporter gene activation (luciferase, β-galactosidase) or by counting syncytia formation.

• Split reporter protein complementation: Cells expressing the viral glycoproteins carry one fragment of a split reporter protein (like GFP or luciferase), while receptor-bearing cells express the complementary fragment. Upon fusion, the reporter fragments combine to produce measurable signal.

• Content mixing assays: Using fluorescent dyes that are activated or quenched upon mixing of cytoplasmic contents following membrane fusion.

• Virus entry assays: Pseudotyping vesicular stomatitis virus (VSV) or lentiviral particles with recombinant SaHV-2 gB (along with other required glycoproteins) and measuring entry into permissive cells.

Since gB interacts with other viral glycoproteins during fusion, these assays should include controls testing the requirement for gD and gH/gL heterodimers, which form the minimum fusion complex with gB .

How can researchers accurately assess the binding of SaHV-2 gB to cellular receptors?

Receptor binding studies for SaHV-2 gB should consider known receptor interactions from related alphaherpesviruses. Based on HSV-1 studies, gB can directly interact with cellular receptors including the non-muscle myosin heavy chain (NMHC-IIA) and paired immunoglobulin-like type 2 receptor α (PILR-α) .

The following methodologies are recommended for comprehensive receptor binding analysis:

• Surface plasmon resonance (SPR): Provides real-time binding kinetics between purified recombinant gB and soluble receptor proteins or peptides

• Co-immunoprecipitation: Identifies interactions between gB and cellular proteins in a more native context

• Fluorescence resonance energy transfer (FRET): Measures proximity-based interactions between tagged gB and receptor proteins

• Cross-linking studies: Captures transient interactions between gB and cellular binding partners

Researchers should also explore whether the amino terminus of SaHV-2 gB shares similar receptor binding properties as HSV-1 gB, particularly since the amino terminus has been shown to be critical for PILR-α receptor binding, which influences viral pathogenicity .

What key structural and functional domains of SaHV-2 gB can be targeted for comparative mutagenesis studies?

Based on knowledge of herpesvirus gB structure-function relationships, several domains represent prime targets for comparative mutagenesis:

• Fusion loops: These hydrophobic regions insert into target membranes and are critical for the fusion process. Comparing fusion loop sequences between SaHV-2 and other primate herpesvirus gB proteins could reveal adaptations to specific host cell membranes.

• Receptor binding domains: The amino terminus of gB has been implicated in receptor binding, particularly to PILR-α . Mutations in this region could help map species-specific receptor interactions.

• Glycosylation sites: N-linked and O-linked glycosylation sites influence protein folding, immune evasion, and potentially receptor interactions . Comparative glycosylation site mutagenesis could reveal their functional significance.

• gH/gL interaction interfaces: Regions of gB that interact with the gH/gL heterodimer during fusion activation are critical for coordinated fusion. These sites may show species-specific adaptations.

• Trimeric interface residues: Mutations at the interfaces between protomers in the gB trimer could reveal differences in stability and activation thresholds.

Targeted mutagenesis studies comparing these domains between SaHV-2 gB and other primate herpesvirus gB proteins would provide valuable insights into functional divergence and potentially identify determinants of host range and pathogenicity.

What approaches can researchers use to identify neutralizing epitopes in SaHV-2 gB?

Identifying neutralizing epitopes in SaHV-2 gB requires a systematic approach combining computational predictions with experimental validation:

• Epitope mapping using overlapping peptide arrays: Synthesize overlapping peptides spanning the entire SaHV-2 gB sequence and test their reactivity with neutralizing antibodies obtained from infected or immunized animals.

• Phage display libraries: Screen phage-displayed peptide libraries with neutralizing antibodies to identify mimotopes that may correspond to conformational epitopes.

• Computational prediction: Use structure-based epitope prediction algorithms to identify surface-exposed regions likely to serve as antibody binding sites, particularly focusing on regions that undergo conformational changes during fusion.

• Competitive binding assays: Determine whether antibodies compete for binding to recombinant gB, suggesting spatial proximity of their epitopes.

• Escape mutant analysis: Generate viral escape mutants under selective pressure from neutralizing antibodies and sequence the gB gene to identify mutations that confer resistance.

Based on studies of HSV gB, researchers should pay particular attention to domains involved in receptor binding and fusion, as these often harbor important neutralizing epitopes . The amino terminus of gB, which interacts with cellular receptors like PILR-α, represents a promising region for neutralizing epitope identification .

How can cross-reactivity studies between SaHV-2 gB and other herpesvirus gB proteins inform vaccine design?

Cross-reactivity studies between SaHV-2 gB and other herpesvirus gB proteins can provide crucial insights for vaccine design, particularly for developing broadly protective vaccines:

• Antibody cross-reactivity assessment: Test whether antibodies raised against SaHV-2 gB can neutralize other primate herpesviruses and vice versa. This requires:

  • Pseudotyped virus neutralization assays

  • ELISA binding studies with recombinant proteins

  • Western blot analysis under reducing and non-reducing conditions

• T-cell epitope conservation analysis: Identify T-cell epitopes in SaHV-2 gB and assess their conservation across other herpesviruses using:

  • Peptide-MHC binding prediction algorithms

  • In vitro T-cell stimulation assays with overlapping peptides

  • Adoptive transfer studies in appropriate animal models

• Structural mapping of conserved epitopes: Map identified cross-reactive epitopes onto predicted or experimentally determined structures of SaHV-2 gB to understand their accessibility and conformational requirements.

These cross-reactivity studies can inform rational design of chimeric immunogens incorporating the most conserved neutralizing epitopes from multiple herpesvirus gB proteins. For example, the HSV-1 live-attenuated vaccine strain VC2, which includes deletions affecting the fusion complex, produces robust immune responses compared to its parental strain . Similar approaches could be explored for SaHV-2 gB-based immunogens.

What strategies should researchers employ to generate recombinant SaHV-2 expressing modified gB proteins?

Generating recombinant SaHV-2 with modified gB proteins requires careful consideration of both molecular cloning strategies and functional implications:

• Bacterial Artificial Chromosome (BAC) cloning: The most versatile approach involves maintaining the viral genome as a BAC, allowing precise genetic manipulation:

  • Use two-step Red recombination for scarless mutagenesis

  • Design homology arms of 40-50 bp flanking the gB gene

  • Include selection markers (kanamycin, chloramphenicol) for initial selection

  • Use counter-selection (SacB, I-SceI) for marker removal

• CRISPR/Cas9-assisted homologous recombination: For labs without established BAC systems:

  • Design guide RNAs targeting sequences flanking the gB gene

  • Create repair templates containing modified gB with homology arms

  • Co-transfect guide RNAs, Cas9, and repair template into infected cells

  • Screen recombinant viruses by PCR and sequencing

• Preservation of critical functional domains: When designing modifications, researchers must preserve domains essential for:

  • Proper folding and trimerization

  • Interactions with other glycoproteins in the fusion complex

  • Receptor binding capabilities

• Validation of recombinant viruses: Comprehensive characterization should include:

  • Growth curve analysis in multiple cell types

  • Plaque morphology assessment

  • Fusion assays comparing wild-type and modified gB

  • Western blot verification of gB expression levels and processing

When designing modifications, researchers should consider the findings from HSV-1 studies showing that deletions in gK glycosylation sites affect the fusion complex functionality and can result in attenuated viruses with potential vaccine applications .

How can researchers design chimeric gB proteins between SaHV-2 and other herpesviruses to study structure-function relationships?

Designing chimeric gB proteins between SaHV-2 and other herpesviruses provides powerful tools for dissecting domain-specific functions:

• Domain swapping strategy: Based on predicted structural domains, researchers can design chimeras that replace specific functional regions:

  • Fusion loops responsible for membrane insertion

  • Receptor binding domains, particularly the amino terminus

  • Central domain involved in trimerization

  • gH/gL interaction interfaces

  • Transmembrane and cytoplasmic domains

• Fusion junction design considerations:

  • Place fusion junctions in structurally conserved regions to minimize disruption

  • Maintain secondary structure elements (avoid breaking alpha-helices or beta-sheets)

  • Consider using flexible linkers at domain boundaries if necessary

  • Verify that chimeric constructs maintain correct disulfide bonding patterns

• Experimental validation approach:

  • Express chimeric proteins in mammalian cells

  • Assess proper folding and trimerization using size exclusion chromatography

  • Test fusion function using cell-cell fusion assays

  • Evaluate receptor binding using SPR or cell binding assays

This chimeric approach can be particularly informative given previous findings that gB homologs from HSV-1 and SaHV-1 were interchangeable in functional assays, while other glycoproteins like gD and gH/gL were not interchangeable between these viruses . This suggests that despite high conservation, subtle differences in gB may contribute to species-specific functions.

How can recombinant SaHV-2 gB be utilized in the development of novel antiviral strategies?

Recombinant SaHV-2 gB offers several avenues for developing novel antiviral strategies:

• Structure-based drug design:

  • Generate high-resolution structures of SaHV-2 gB in pre-fusion and post-fusion conformations

  • Identify druggable pockets that could be targeted to prevent conformational changes required for fusion

  • Use in silico screening to identify small molecules that bind these pockets

  • Validate candidates using in vitro fusion inhibition assays

• Fusion inhibitor peptides:

  • Design peptides derived from SaHV-2 gB domains involved in critical protein-protein interactions

  • Test their ability to competitively inhibit interactions required for the fusion process

  • Optimize peptide stability, cell penetration, and target binding

• Receptor decoy strategies:

  • Create soluble forms of gB receptor binding domains that compete with cellular receptors

  • Engineer high-affinity decoy receptors that bind gB and prevent its interaction with cellular targets

  • Test whether these approaches inhibit viral entry in cell culture systems

• Broadly reactive monoclonal antibodies:

  • Identify conserved epitopes between SaHV-2 gB and other herpesvirus gB proteins

  • Generate monoclonal antibodies targeting these regions

  • Test their neutralizing capacity against multiple herpesviruses

  • Determine mechanisms of neutralization (blocking receptor binding, preventing conformational changes, etc.)

The growing resistance to current anti-herpesvirus drugs like acyclovir, famciclovir, and valacyclovir, particularly in immunocompromised patients, highlights the need for new antiviral approaches targeting different viral components like gB .

What are the considerations for using SaHV-2 gB in vector-based vaccine platforms?

When developing SaHV-2 gB-based vector vaccines, researchers should consider several critical factors:

• Antigen design optimization:

  • Express full-length gB versus soluble truncated forms lacking the transmembrane domain

  • Evaluate pre-fusion stabilized variants that preserve neutralizing epitopes

  • Consider co-expression with other glycoproteins (gD, gH/gL) to present the complete fusion complex

  • Test mutations that enhance immunogenicity while maintaining proper folding

• Vector platform selection:

  • Viral vectors (Adenovirus, MVA, VSV) for strong cellular responses

  • mRNA platforms for direct protein expression and flexibility in antigen design

  • DNA vaccines for simplicity and stability

  • Virus-like particles displaying gB for enhanced B-cell responses

• Immunological considerations:

  • Balance humoral versus cellular immune responses

  • Target both neutralizing antibody production and T-cell responses

  • Consider adjuvants that enhance specific immune pathways

  • Evaluate prime-boost strategies with heterologous vectors

• Preclinical evaluation metrics:

  • Neutralizing antibody titers against both homologous and heterologous viruses

  • T-cell responses measured by ELISpot and intracellular cytokine staining

  • Protection in appropriate animal challenge models

  • Durability of immune responses over time

Lessons can be drawn from the HSV-1 live-attenuated vaccine strain VC2, which includes deletions of both gK glycosylation sites and UL20, preventing the virion from entering via fusion while still producing robust immune responses . Similar strategies could be applied to SaHV-2 gB-based vaccines, potentially creating attenuated viruses or vector-expressed antigens that stimulate protective immunity while avoiding pathology.

What are the common challenges in maintaining proper conformation of recombinant SaHV-2 gB and how can they be addressed?

Researchers frequently encounter challenges maintaining the native conformation of recombinant herpesvirus glycoproteins, including SaHV-2 gB. Based on experiences with related viral glycoproteins, these challenges and their solutions include:

• Protein aggregation:

  • Problem: Recombinant gB often forms aggregates during expression and purification

  • Solutions:

    • Express at lower temperatures (28-30°C) to slow folding and prevent aggregation

    • Include mild detergents (0.01-0.05% NP-40 or Triton X-100) in purification buffers

    • Add low concentrations (10-15%) of glycerol as a stabilizing agent

    • Optimize pH conditions to maintain stability (typically pH 7.2-7.4)

• Improper glycosylation:

  • Problem: Incorrect or incomplete glycosylation affecting folding and function

  • Solutions:

    • Use mammalian expression systems (HEK293, CHO) rather than insect or bacterial systems

    • Avoid cell lines with altered glycosylation (GnTI- cells) unless studying specific glycoform effects

    • Consider enzymatic deglycosylation tests to determine glycosylation dependence of folding

• Premature triggering of conformational changes:

  • Problem: gB prematurely shifting from pre-fusion to post-fusion conformation

  • Solutions:

    • Introduce stabilizing mutations based on structure prediction

    • Maintain low temperature throughout purification

    • Consider adding fusion peptide inhibitors during purification

    • Use conformation-specific antibodies to monitor conformational state

• Proteolytic degradation:

  • Problem: Susceptibility to proteolysis during expression and purification

  • Solutions:

    • Include protease inhibitor cocktails in all buffers

    • Identify and mutate exposed protease-sensitive sites

    • Optimize purification speed to minimize exposure time

Addressing these challenges requires iterative optimization and validation using functional assays to confirm that the recombinant protein retains native-like properties.

How can researchers optimize transfection conditions for co-expression of SaHV-2 gB with other viral glycoproteins?

Co-expression of multiple herpesvirus glycoproteins presents unique challenges for studying functional complexes. For SaHV-2 gB co-expression with other viral glycoproteins (gD, gH/gL, gK), researchers should consider:

• Vector design considerations:

  • Use vectors with different promoter strengths to balance expression levels

  • Consider multi-cistronic constructs using 2A peptides or IRES elements

  • Test both separate plasmids and single plasmids with multiple expression cassettes

  • Include different selection markers for stable cell line development

• Transfection ratio optimization:

  • Test different ratios of glycoprotein-encoding plasmids (e.g., gB:gD:gH:gL at 1:1:1:1, 2:1:1:1, etc.)

  • Analyze protein expression levels by Western blot to confirm desired ratios

  • Correlate expression ratios with functional outcomes in fusion assays

• Transfection method selection:

  • For transient co-expression: lipid-based reagents (Lipofectamine) or PEI for higher efficiency

  • For stable cell lines: viral vectors or transposon-based systems for consistent co-expression

  • For primary cells: electroporation or nucleofection may provide better efficiency

• Timing considerations:

  • Synchronize expression by using inducible promoters (tetracycline-responsive)

  • Evaluate expression kinetics to determine optimal time points for functional assays

  • Consider sequential transfection if certain glycoproteins affect expression of others

Successful co-expression studies enable researchers to investigate the complex interactions between gB and other glycoproteins that form the viral fusion machinery, particularly the minimum fusion complex of gB, gD, gH, and gL that has been established for related herpesviruses .

How should researchers interpret species-specific differences in SaHV-2 gB function compared to other herpesvirus gB proteins?

Interpreting species-specific differences in SaHV-2 gB requires a multifaceted approach that considers both molecular mechanisms and evolutionary context:

These interpretations should consider that viral glycoproteins evolve under multiple selective pressures, including adaptation to host receptors, immune evasion, and maintenance of essential functions in viral replication.

What methodological approaches can resolve contradictory findings in SaHV-2 gB functional studies?

When researchers encounter contradictory findings in SaHV-2 gB studies, systematic methodological approaches can help resolve these discrepancies:

• Standardization of experimental systems:

  • Develop consensus protocols for expression, purification, and functional assays

  • Create reference standards (antibodies, protein preparations) that can be shared between laboratories

  • Establish validated cell lines that express consistent levels of relevant receptors

• Multi-method validation approach:

  • Apply complementary techniques to test the same hypothesis

  • For entry studies: compare results from virus infectivity, cell-cell fusion, and biochemical binding assays

  • For structural studies: validate findings using multiple biophysical methods (CD spectroscopy, DSC, SPR)

• Contextual variables examination:

  • Systematically test how experimental conditions affect outcomes:

    • Cell type dependencies (receptor expression profiles)

    • Buffer composition effects (pH, ionic strength)

    • Temporal factors (protein stability over time)

    • Temperature dependencies

• Genetic determinant mapping:

  • Use site-directed mutagenesis to precisely map functional determinants

  • Create chimeric constructs to isolate domain-specific effects

  • Apply reverse genetics to validate findings in the context of infectious virus

• Collaborative cross-validation:

  • Organize multi-laboratory studies where identical materials are tested using standardized protocols

  • Compare results across different geographic locations and experimental setups

  • Identify variables that consistently affect experimental outcomes

By implementing these approaches, researchers can distinguish genuine biological variations from methodological artifacts, leading to more robust and reproducible findings in SaHV-2 gB research.

What emerging technologies will advance our understanding of SaHV-2 gB structure and function?

Several cutting-edge technologies are poised to transform our understanding of herpesvirus glycoproteins, including SaHV-2 gB:

• Cryo-electron microscopy advances:

  • Single-particle cryo-EM at near-atomic resolution can capture different conformational states of gB

  • Cryo-electron tomography can visualize gB in the context of the viral envelope

  • Time-resolved cryo-EM may eventually capture fusion intermediates

• Integrative structural biology approaches:

  • Combining X-ray crystallography, NMR, and SAXS data for comprehensive structural models

  • Mass spectrometry with hydrogen-deuterium exchange to map conformational dynamics

  • Computational approaches like AlphaFold2 to predict structures of poorly characterized domains

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize gB distribution and clustering during entry

  • Single-molecule FRET to track conformational changes in real-time

  • Correlative light and electron microscopy to connect functional events with ultrastructural changes

• Genome editing technologies:

  • CRISPR-Cas base editing for precise amino acid substitutions without double-strand breaks

  • Prime editing for targeted sequence replacements in the viral genome

  • High-throughput screening of gB variants using CRISPR libraries

• Systems biology approaches:

  • Proteomics to identify the complete interactome of SaHV-2 gB

  • Transcriptomics to understand cellular responses to gB expression

  • Mathematical modeling of the fusion process incorporating multiple glycoproteins

These technologies will help address fundamental questions about how gB mediates membrane fusion in concert with other viral glycoproteins and how these processes are regulated in different cellular contexts.

How might research on SaHV-2 gB contribute to broader understanding of viral entry mechanisms and host-pathogen interactions?

Research on SaHV-2 gB has significant potential to advance our understanding of viral entry mechanisms and host-pathogen interactions in several ways:

• Comparative virology insights:

  • Studying SaHV-2 gB alongside other primate herpesvirus gB proteins can reveal conserved versus divergent strategies for host cell entry

  • Identification of species-specific adaptations may explain differences in host range and pathogenicity

  • Understanding how viruses from different hosts have evolved similar entry mechanisms provides insight into convergent evolution

• Fundamental membrane fusion mechanisms:

  • The complex regulation of herpesvirus fusion involving multiple glycoproteins serves as a model for understanding protein-mediated membrane fusion

  • Insights from SaHV-2 gB may be applicable to other class III fusion proteins from diverse virus families

  • Mechanistic details could inform understanding of cellular fusion processes that use similar protein machinery

• Host-pathogen co-evolution:

  • Analysis of SaHV-2 gB receptor interactions can reveal how viruses adapt to specific host factors

  • Comparative studies across primate species may identify genetic determinants of susceptibility or resistance

  • Evidence of recombination events among herpesviruses suggests mechanisms for rapid adaptation to new hosts

• Translational applications:

  • Identification of conserved mechanisms across herpesvirus gB proteins could lead to broadly effective antiviral strategies

  • Understanding species-specific differences may explain why some antiviral approaches fail when applied across different viruses

  • Novel vaccine strategies for human herpesviruses may emerge from comparative studies with SaHV-2

• Model systems development:

  • SaHV-2 could serve as a safer model system for studying aspects of more pathogenic herpesviruses

  • Recombinant viruses expressing chimeric gB proteins could help identify determinants of neurotropism and virulence

By pursuing these research directions, studies of SaHV-2 gB will contribute not only to our understanding of this specific virus but also to broader principles of viral entry, evolution, and host-pathogen interactions.