Recombinant Adelaide River virus Glycoprotein G (G)

How does ARV Glycoprotein G compare functionally to similar proteins in other viruses?

ARV Glycoprotein G belongs to the family of rhabdovirus attachment proteins and shares functional characteristics with other viral glycoproteins while maintaining its unique properties:

Like other rhabdovirus G proteins, ARV Glycoprotein G associates into trimers to form viral surface projections that bind to host cell receptors . It induces virus endocytosis and mediates fusion between viral and endosomal membranes . The protein is involved in viral tropism and pathogenicity, and it induces and binds virus-neutralizing antibodies while eliciting cell-mediated immune responses .

In contrast to HIV envelope glycoproteins like gp120, which have five conserved regions (C1-C5) and five variable regions (V1-V5) with up to 60-80% sequence variability, ARV G protein demonstrates higher sequence conservation within its family .

What is the evolutionary relationship between ARV G and GNS glycoproteins?

The ARV genome contains two adjacent glycoprotein genes encoding the G protein and a non-structural glycoprotein (GNS) . Multiple sequence alignments and phylogenetic analyses indicate that the ARV G and GNS glycoproteins are structurally related and appear to have evolved at different rates from a common ancestral gene .

The evolutionary relationship between these proteins reveals an interesting example of gene duplication as an evolutionary process. The second ORF in the ARV genome encodes a GNS polypeptide of 609 residues with nine potential glycosylation sites . This protein is most closely related to the BEFV non-structural glycoprotein .

A proposed evolutionary mechanism involves a copy-choice process during viral replication:

• A copy-choice mechanism involving upstream relocation of the polymerase during replication may account for the evolution of these tandem glycoprotein genes

• This mechanism allows for gene duplication followed by divergent evolution

• The differential evolutionary rates have resulted in proteins with distinct but related functions

Phylogenetic analysis of the ARV nucleoprotein (N) gene places ARV and BEFV as closely related viruses (48.3% similarity) that share higher sequence similarity to vesiculoviruses than to lyssaviruses . This positioning in the phylogenetic tree is consistent with the genome organization and host range of these viruses .

What expression systems are optimal for producing functional recombinant ARV Glycoprotein G?

The choice of expression system for recombinant ARV Glycoprotein G production depends on research objectives, especially regarding post-translational modifications and functional studies:

For E. coli expression, the full-length ARV Glycoprotein G can be produced with an N-terminal His tag for easy purification . The protein (amino acids 23-660) can be expressed and purified using metal affinity chromatography . After purification, it's recommended to store the protein as a lyophilized powder and reconstitute it in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage at -20°C/-80°C .

For mammalian expression systems, researchers should consider using HEK293 cells, similar to the approach used for RSV glycoprotein production . This system allows proper glycosylation and folding that more closely resembles the native viral protein. A construct design including a C-terminal tag (such as human Fc) with a glycine-serine linker can facilitate purification while maintaining protein functionality .

When selecting an expression system, researchers should consider:

• The requirement for proper glycosylation (6 N-linked sites in ARV G)

• The intended downstream applications (structural vs. functional studies)

• The need for proper folding and oligomerization (trimeric structure)

• Required yield and scale of production

What methodologies are most effective for studying ARV Glycoprotein G interactions with host cell receptors?

Investigating ARV Glycoprotein G interactions with host cell receptors requires a combination of molecular, biochemical, and cell-based techniques:

• Receptor Identification Approaches:

  • Virus overlay protein binding assay (VOPBA) to identify cellular proteins that interact with purified G protein

  • Co-immunoprecipitation followed by mass spectrometry to identify binding partners

  • CRISPR-Cas9 knockout screens to identify essential host factors

  • Comparative analysis with BEFV G protein receptor interactions given their sequence homology

• Binding Kinetics and Affinity Measurements:

  • Surface plasmon resonance (SPR) using recombinant G protein and purified receptor candidates

  • Bio-layer interferometry (BLI) for label-free real-time binding analysis

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

• Functional Assays:

  • Cell-cell fusion assays using G protein-expressing cells and receptor-expressing cells

  • Pseudotyped virus entry assays incorporating ARV G protein into pseudovirus particles

  • Site-directed mutagenesis of key residues followed by binding and fusion assays

• Structural Studies:

  • Cryo-electron microscopy of G protein-receptor complexes

  • X-ray crystallography of G protein domains with receptor fragments

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map binding interfaces

When designing receptor interaction studies, researchers should consider that rhabdovirus G proteins typically bind to host cell receptors, induce virus endocytosis, and mediate fusion of viral and endosomal membranes . The involvement of ARV G protein in tropism and pathogenicity suggests that receptor specificity plays a crucial role in viral infection dynamics .

A methodological workflow might involve:

• Initial screening for binding partners using pull-down assays or crosslinking approaches

• Validation of specific interactions using recombinant proteins and quantitative binding assays

• Functional confirmation through cell-based entry assays

• Detailed mapping of binding interfaces through mutagenesis and structural studies

How can post-translational modifications of ARV Glycoprotein G be analyzed in different expression systems?

Post-translational modifications (PTMs) of ARV Glycoprotein G, particularly the six potential N-linked glycosylation sites , are critical for proper protein folding, function, and immunogenicity. Comprehensive analysis of these modifications requires multiple complementary techniques:

1. Glycosylation Site Mapping:

• Site-directed mutagenesis of predicted N-glycosylation sites (Asn-X-Ser/Thr)

• Mass spectrometry analysis:

  • Peptide mass fingerprinting after proteolytic digestion

  • Electron transfer dissociation (ETD) to preserve glycan structures

  • Hydrophilic interaction liquid chromatography (HILIC) for glycopeptide enrichment

2. Glycan Composition Analysis:

• Release of N-glycans using PNGase F treatment

• Permethylation followed by MALDI-TOF MS analysis

• High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

• Comparison of glycan profiles between expression systems:

3. Functional Impact Assessment:

• Comparative receptor binding assays of differentially glycosylated forms

• Neutralization assays with glycoform-specific antibodies

• Cell fusion assays to assess the impact of glycosylation on membrane fusion

4. Other PTM Analyses:

• Palmitoylation analysis using click chemistry with alkyne-tagged palmitic acid

• Phosphorylation site mapping using phospho-enrichment techniques

• Disulfide bond mapping using non-reducing/reducing gel comparisons and MS analysis

5. Integrated Structural Analysis:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to assess structural impacts of glycosylation

• Small-angle X-ray scattering (SAXS) to determine solution structures of different glycoforms

• Molecular dynamics simulations incorporating glycan structures

Researchers should consider the biological relevance of the expression system. For functional studies, mammalian expression systems like HEK293 would provide glycosylation patterns more similar to those in the native virus . For structural studies where glycan heterogeneity might hinder crystallization, controlled deglycosylation or expression of protein fragments in E. coli might be advantageous .

What approaches can address challenges in crystallizing recombinant ARV Glycoprotein G for structural studies?

Crystallizing viral envelope glycoproteins like ARV Glycoprotein G presents significant challenges due to their flexibility, glycosylation heterogeneity, and tendency to form trimeric assemblies. The following methodological approaches can help researchers overcome these obstacles:

1. Protein Engineering Strategies:

• Core domain construction: Remove highly flexible regions while maintaining structural integrity

• Surface entropy reduction: Replace surface clusters of high-entropy residues (Lys, Glu) with alanines

• Fusion partners: Use crystallization chaperones like T4 lysozyme or BRIL

• Disulfide engineering: Introduce disulfide bonds to stabilize flexible regions

2. Glycan Management:

• Expression in GnTI-deficient cell lines to produce homogeneous high-mannose glycans

• Enzymatic deglycosylation using EndoH or PNGase F under native conditions

• Site-directed mutagenesis to eliminate non-essential glycosylation sites

• Co-crystallization with deglycosylation enzymes

3. Crystallization Condition Optimization:

• High-throughput screening using sparse matrix approaches

• Inclusion of specific additives known to facilitate membrane protein crystallization

• Lipidic cubic phase crystallization for capturing membrane-proximal regions

• Silver bullet screens to identify stabilizing small molecules

4. Alternative Stabilization Approaches:

• Co-crystallization with neutralizing antibody Fab fragments

• Complex formation with host receptor fragments

• Stabilization in the pre- or post-fusion conformations using specific mutations

5. Complementary Structural Techniques:

• Cryo-electron microscopy for full-length protein structure

• Small-angle X-ray scattering (SAXS) for solution structure

• Nuclear magnetic resonance (NMR) for specific domains

Drawing from experiences with HIV envelope glycoprotein structural studies , researchers should consider:

• The successful crystallization of HIV gp120 core with CD4 and antibody fragments at 2.2 Å resolution

• The structural determination of glycosylated unliganded SIV gp120 core at 4 Å

• The recent crystal structure of JR-FL gp120 core with V3 in complex with CD4 and Fab X5 at 3.5 Å

These examples suggest that complexing ARV Glycoprotein G with binding partners and removing highly flexible regions while preserving key functional domains may facilitate crystallization. Researchers should plan for multiple constructs and crystallization strategies in parallel to maximize chances of success.

What are the best approaches for analyzing ARV Glycoprotein G antigenic properties and immunogenicity?

Comprehensive analysis of ARV Glycoprotein G antigenic properties and immunogenicity requires a multifaceted approach combining in vitro, in silico, and in vivo methods:

1. Epitope Mapping Techniques:

• Peptide scanning: Overlapping peptide arrays covering the entire sequence

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify antibody binding footprints

• X-ray crystallography or cryo-EM of antibody-antigen complexes

• Phage display for conformational epitope mapping

• Computational epitope prediction and validation

2. Neutralizing Antibody Characterization:

• Pseudovirus neutralization assays

• Antibody-dependent cellular cytotoxicity (ADCC) assays

• Complement-dependent cytotoxicity (CDC) assays

• Surface plasmon resonance (SPR) for binding kinetics and affinity measurements

• Competition binding assays to define epitope clusters

3. T-cell Response Analysis:

• ELISPOT assays for IFN-γ, IL-2, or other cytokines

• Intracellular cytokine staining and flow cytometry

• T-cell proliferation assays

• HLA binding prediction and validation

• TCR repertoire analysis after immunization

4. Comparative Immunogenicity Assessment:

• Comparison of different immunogens:

5. In Vivo Evaluation Methods:

• Animal model selection based on receptor compatibility

• Prime-boost immunization strategies

• Challenge studies to assess protection

• Passive transfer experiments with purified antibodies

• Long-term immunity and memory response assessment

6. Structure-Based Immunogen Design:

• Stabilization of prefusion conformations

• Glycan engineering to expose neutralizing epitopes

• Germline-targeting immunogen design

• Sequential immunization with evolutionarily guided immunogens

When analyzing ARV Glycoprotein G immunogenicity, researchers should consider that the protein induces and binds virus-neutralizing antibodies and elicits cell-mediated immune responses . Comparative studies with BEFV G protein might provide valuable insights given their sequence homology .

Researchers should also consider the structure-function relationships revealed in HIV Env studies , where crystallographic structures have informed immunogen design. Similar approaches could be applied to ARV Glycoprotein G, particularly focusing on conserved functional domains that might elicit broadly neutralizing antibodies.