Recombinant Varicella-zoster virus Virion egress protein 24 (24)

What is the structural composition of the VZV ORF24-ORF27 nuclear egress complex?

The VZV ORF24-ORF27 complex forms a critical heterodimeric structure essential for viral nuclear egress. The crystal structure has been determined at 2.1 Å resolution, revealing that ORF24 functions as a "groove protein" that interacts with ORF27, which serves as a "hook protein" . Individually purified ORF24 and ORF27 proteins behave as monomers in solution but readily form a stable heterodimeric complex upon mixing . This structural arrangement is similar to that observed in other alpha-herpesviruses, though with distinct subfamily-specific features.

Methodologically, researchers have successfully characterized this complex through:

• X-ray crystallography for high-resolution structural determination

• Solution-based binding assays to assess protein-protein interactions

• Comparative structural analysis with other herpesvirus NECs

How does ORF24 interact with other viral and cellular proteins during infection?

To study these interactions, researchers should employ:

• Coimmunoprecipitation (CoIP) assays

• Confocal laser scanning microscopy (CLSM) for colocalization studies

• Yeast two-hybrid screens for identifying novel interaction partners

• GST pull-down assays to validate direct protein-protein interactions

What expression systems are most effective for producing recombinant ORF24?

Based on established protocols for VZV proteins, several expression systems can be employed for recombinant ORF24 production:

Bacterial Expression Systems:

• BL21(DE3) and Rosetta DE3 cells have been successfully used for bacterial expression of 6×His- or MBP-tagged VZV proteins .

• A typical protocol involves:

  • Transforming expression vectors encoding VZV ORFs into bacterial cells

  • Growing cultures in selective LB medium (50 μg/ml of ampicillin, 50 μg/ml of chloramphenicol)

  • Inducing protein expression at optimal OD600

  • Harvesting and lysing cells, followed by affinity purification

Mammalian Cell Systems:
For functional studies in a more native context, mammalian expression is preferable, using:

• Vectors with strong promoters (CMV) for transient expression

• Stable cell lines for consistent protein production

• Fusion tags that facilitate detection and purification while minimizing interference with protein function

What are the homologous relationships between VZV ORF24 and other herpesvirus proteins?

VZV ORF24 is homologous to proteins in other herpesviruses, exhibiting subfamily-specific conservation patterns:

This homology pattern explains the experimental finding that VZV ORF24 can interact with HSV-1 NEC proteins but not with those from beta or gamma herpesviruses . The capacity of VZV NEC proteins to undergo heterodimeric interactions appears to be restricted within proteins of the α-herpesviral subfamily.

What methodological approaches can resolve the functional domains of ORF24?

To characterize functional domains within ORF24, researchers should implement a multi-faceted approach:

Mutational Analysis Strategy:

• Generate alanine-scanning mutants across conserved regions

• Create deletion constructs targeting predicted functional domains

• Employ site-directed mutagenesis for residues identified by computational alanine scanning

• Test mutants for:

  • Ability to form complexes with ORF27 (by CoIP)

  • Nuclear localization (by immunofluorescence)

  • Functional complementation in ORF24-deficient viral backgrounds

Domain Mapping Protocol:

• Express truncated versions of ORF24 as fusion proteins

• Assess interaction with full-length ORF27 and other potential partners

• Complement findings with hydrogen-deuterium exchange mass spectrometry to identify solvent-exposed regions

Computational alanine scanning of the ORF24-ORF27 interface has already revealed several shared intermolecular interaction features that differ from β/γ-herpesviral NECs , providing valuable starting points for experimental validation.

How can recombineering approaches be utilized to study ORF24 function in the viral context?

Recombineering represents a powerful approach for studying ORF24 function within the viral genome context:

Recombineering Protocol:

• Generate a bacterial artificial chromosome (BAC) containing the VZV genome with a deletion or modification in the ORF24 gene

• Prepare overlapping DNA fragments containing desired mutations using high-fidelity PCR

• Transform these fragments into cells containing the BAC for homologous recombination

• Select recombinants using appropriate markers (such as DOG plates)

• Screen colonies for successful recombination by PCR and sequencing

• Transfect confirmed BAC constructs into mammalian cells to generate mutant viruses

This approach has been successfully implemented for VZV ORF54 mutants, where single nucleotide changes were introduced that conferred resistance to specific inhibitors . A similar methodology could be applied to ORF24 to:

• Generate point mutations in functional domains

• Create tagged versions for visualization

• Develop conditional expression systems to study temporal requirements

What techniques are most effective for analyzing ORF24-ORF27 complex formation kinetics?

Understanding the assembly kinetics of the ORF24-ORF27 complex requires specialized biophysical approaches:

Recommended Techniques:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified ORF24 on a sensor chip

  • Flow varying concentrations of ORF27 over the surface

  • Measure association and dissociation rates

  • Calculate binding affinity constants

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding

  • Determine stoichiometry, binding constants, and energetics

  • No protein labeling or immobilization required

• Microscale Thermophoresis (MST):

  • Label one protein partner with a fluorescent dye

  • Measure changes in thermophoretic mobility upon binding

  • Calculate binding parameters in solution

Studies with related herpesvirus NECs have shown nanomolar affinities between NEC components , suggesting similar high-affinity interactions may exist between ORF24 and ORF27.

How can structural information about the ORF24-ORF27 complex inform antiviral drug development?

The 2.1 Å resolution crystal structure of the VZV ORF24-ORF27 complex provides an excellent foundation for structure-based drug design:

Drug Development Methodology:

• Identify "hotspot" residues at the ORF24-ORF27 interface through computational analysis

• Target protein-protein interaction surfaces unique to the herpesvirus subfamily

• Perform virtual screening of compound libraries against identified pockets

• Test hit compounds for:

  • Binding to purified ORF24 (thermal shift assays, SPR)

  • Disruption of ORF24-ORF27 complex formation (FRET-based assays)

  • Inhibition of viral replication in cell culture

  • Specificity against alpha-herpesviruses versus other subfamilies

The structural comparison revealing differences between alpha-herpesviral NECs versus beta/gamma-herpesviral NECs suggests opportunities for developing subfamily-specific antiviral compounds that disrupt the ORF24-ORF27 interaction.

What approaches can differentiate the role of ORF24 phosphorylation in nuclear egress?

Phosphorylation often regulates nuclear egress complex function in herpesviruses:

Experimental Approach:

• Identify potential phosphorylation sites in ORF24 using:

  • Mass spectrometry of purified protein from infected cells

  • Phosphorylation-specific antibodies

  • In silico prediction tools

• Generate phosphomimetic (S/T→D/E) and phosphodeficient (S/T→A) mutants

• Analyze mutant phenotypes:

  • Localization by immunofluorescence

  • Complex formation with ORF27

  • Viral replication kinetics

  • Virion production and morphology by electron microscopy

• Employ phosphorylation state-specific antibodies to track temporal changes during infection cycle

For cellular lysate preparation, RIPA buffer containing protease inhibitors has been successfully used for VZV protein analysis , and could be supplemented with phosphatase inhibitors for phosphorylation studies.

How can viral and recombinant ORF24 protein purification be optimized?

Purification Strategy Options:

• His-tagged ORF24 Purification:

  • Transform expression vectors into BL21(DE3) or Rosetta DE3 cells

  • Grow in selective LB medium (50 μg/ml ampicillin, 50 μg/ml chloramphenicol)

  • Induce expression at optimal OD600

  • Harvest cells and purify using nickel affinity chromatography

  • Further purify by size exclusion chromatography to ensure monomeric state

• Native ORF24 from Infected Cells:

  • Infect appropriate cell lines (e.g., MeWo cells for VZV)

  • Harvest cells at optimal time post-infection

  • Prepare lysates using RIPA buffer with protease inhibitors

  • Immunoprecipitate ORF24 using specific antibodies

  • Analyze by SDS-PAGE and Western blotting

Key considerations include maintaining protein solubility, preserving native conformation, and ensuring removal of contaminating proteins and nucleic acids.

What methods are most reliable for tracking ORF24 localization during infection?

Localization Analysis Protocol:

• Immunofluorescence Microscopy:

  • Infect cells grown on coverslips with VZV

  • Fix cells at various time points post-infection

  • Permeabilize and block non-specific binding

  • Incubate with primary antibodies against ORF24 and cellular markers

  • Apply fluorescently-labeled secondary antibodies

  • Counterstain nuclei with DAPI

  • Analyze by confocal microscopy to determine subcellular localization

• Live Cell Imaging:

  • Generate recombinant VZV expressing fluorescently tagged ORF24

  • Infect cells and perform time-lapse imaging

  • Track dynamic localization throughout infection

  • Correlate with markers for different cellular compartments

• Biochemical Fractionation:

  • Separate infected cells into nuclear, cytoplasmic, and membrane fractions

  • Analyze fractions by Western blot to quantify ORF24 distribution

  • Compare results with other viral proteins from different gene classes

For validation of proper protein expression, Western blotting techniques using ECL detection have proven effective for analyzing VZV proteins .

How can RNA-seq approaches contribute to understanding ORF24 expression dynamics?

RNA-seq analysis can provide valuable insights into ORF24 expression patterns during infection:

RNA-seq Experimental Design:

• Sample Preparation:

  • Infect relevant cell types with VZV

  • Harvest cells at multiple timepoints post-infection

  • Extract total RNA using established protocols

  • Prepare libraries for RNA-seq

• Data Analysis Workflow:

  • Map reads to the VZV genome

  • Quantify expression using RPKM values

  • Create coverage plots of viral RNA-seq reads

  • Perform differential expression analysis across timepoints

• Validation:

  • Confirm key findings by qPCR of ORF24 and related genes

  • Normalize to appropriate housekeeping genes

  • Compare expression patterns with immediate-early, early, and late viral genes

RNA-seq analysis has successfully been used to study VZV gene expression in different contexts, showing that differentiation state can affect viral gene transcription . Similar approaches could elucidate how ORF24 expression is regulated during infection.

What are promising approaches for investigating ORF24 interactions with host immune factors?

The interaction between VZV proteins and host immunity represents an important research avenue:

Research Strategy:

• T-cell Response Analysis:

  • Identify potential T-cell epitopes within ORF24 using prediction algorithms

  • Test epitope recognition using PBMC from VZV-immune donors

  • Employ ELISpot assays to measure interferon-γ responses

  • Determine if ORF24 contributes to cellular immunity like glycoproteins B and E

• Innate Immunity Interactions:

  • Screen for ORF24 interactions with pattern recognition receptors

  • Assess impact on interferon signaling pathways

  • Measure changes in inflammatory cytokine production

  • Determine whether ORF24 functions in immune evasion

• Host Protein Interaction Network:

  • Perform immunoprecipitation followed by mass spectrometry to identify host binding partners

  • Validate interactions by reciprocal co-IP and functional assays

  • Map interaction domains through mutagenesis

  • Compare results with homologous proteins from other herpesviruses

How might CRISPR/Cas9 genome editing advance ORF24 functional studies?

CRISPR/Cas9 technology offers powerful approaches for studying ORF24:

CRISPR Application Strategy:

• Viral Genome Editing:

  • Design guide RNAs targeting ORF24 in the viral genome

  • Generate defined mutations or deletions

  • Create reporter-tagged versions of ORF24

  • Analyze phenotypic consequences in replication assays

• Host Factor Identification:

  • Conduct genome-wide CRISPR screens to identify host factors required for ORF24 function

  • Create knockout cell lines for validated hits

  • Assess impact on ORF24 localization, complex formation, and viral replication

  • Complement with proteomics to build comprehensive interaction networks

• Temporal Control Systems:

  • Develop inducible CRISPR systems for conditional disruption of ORF24

  • Apply at different stages of viral replication

  • Determine time-sensitive requirements for ORF24 function

This approach can overcome limitations of traditional recombineering methods by enabling rapid generation of multiple viral mutants and host cell manipulations.

What comparative analyses between ORF24 and its homologs might reveal new functional insights?

Comparative studies offer valuable perspectives on ORF24 function:

Comparative Analysis Framework:

Current research has already demonstrated that VZV ORF24 can interact with HSV-1 NEC proteins but not with beta or gamma herpesvirus components . Expanding these studies could reveal evolutionary adaptations in nuclear egress mechanisms across the herpesvirus family.