Recombinant Chandipura virus Glycoprotein G (G)

What is Chandipura virus Glycoprotein G and what is its role in viral infection?

Chandipura virus Glycoprotein G (CHAV-G) is a surface protein of Chandipura virus (CHPV), an emerging human pathogen belonging to the Rhabdoviridae family and Vesiculovirus genus that causes deadly encephalitis, particularly among children in India . CHAV-G serves dual critical functions in the viral life cycle: receptor recognition on host cells and fusion of viral and cellular membranes .

The infection process begins when CHAV-G binds to specific receptors on the host cell surface, facilitating endocytosis of the virion . Following internalization into endosomes, the acidic environment triggers conformational changes in CHAV-G, exposing fusion peptides that mediate fusion between viral and endosomal membranes . This pH-dependent fusion process, occurring below pH 6.5, enables the release of the viral nucleocapsid into the cytoplasm for subsequent stages of infection .

CHAV-G exists in at least two distinct conformational states: a pre-fusion state at neutral pH and a post-fusion state that forms at low pH. This conformational flexibility is essential for its function in membrane fusion and viral entry . Additionally, CHAV-G represents a major antigenic determinant, making it a critical target for vaccine development efforts .

How is recombinant Chandipura virus Glycoprotein G produced for research purposes?

Recombinant Chandipura virus Glycoprotein G (rGp) can be produced using several expression systems, with selection based on specific research requirements:

Baculovirus Expression System:
This is the most commonly reported method for rGp production, as described in vaccine development studies . The process involves:

• Cloning the G-gene of CHPV into a baculovirus transfer vector

• Co-transfecting insect cells with the recombinant vector and linearized baculovirus DNA

• Selecting and propagating recombinant baculoviruses expressing CHPV G

• Scaling up production in insect cell cultures

• Purifying the recombinant G protein using HPLC

Bacterial Expression System:
Alternative approaches use bacterial systems with various tags to facilitate purification:

• Cloning the G gene into bacterial expression vectors with GST, His, or MBP tags

• Transforming into E. coli expression strains

• Optimizing induction conditions including inducer concentration and growth parameters

• Extracting and solubilizing the protein using specific conditions

• Purifying using affinity chromatography based on the fusion tag

For structural studies, researchers have also produced a soluble form of CHAV-G ectodomain (CHAV-Gth) by thermolysin limited-proteolysis of recombinant VSV particles in which the G gene was replaced by that of CHAV . This approach generates a truncated but structurally representative form of the glycoprotein suitable for crystallographic studies.

Quality control typically involves SDS-PAGE, Western blotting, and functional assays to confirm proper folding and biological activity of the purified protein.

What structural characteristics define Chandipura virus Glycoprotein G?

The structural characteristics of Chandipura virus Glycoprotein G have been elucidated through crystallographic studies, particularly of its post-fusion conformation determined at 3.6Å resolution :

Domain Organization:
CHAV-G consists of three distinct domains:

• A fusion domain containing fusion peptides

• A pleckstrin homology domain (PHD)

• A trimerization domain

Conformational States:
The glycoprotein exists in at least two major conformational states:

• Pre-fusion state (at neutral pH): Required for receptor binding

• Post-fusion state (at low pH below 6.5): Forms during the fusion process

The pre-fusion trimer is not stable in solution, and the low-pH-induced membrane association is reversible, which is an unusual property among viral fusion proteins .

Key Structural Features:

The crystal structure revealed that while CHAV-G shares high structural similarity with VSV-G (the prototype vesiculovirus), it has evolved alternate structural solutions in hinge regions between PH and fusion domains, as well as distinct pH-sensitive switches .

How does the intrinsic disorder in Glycoprotein G contribute to its function?

Intrinsic disorder is an important structural feature in Chandipura virus proteins, including Glycoprotein G. Analysis of the "dark proteome" of CHPV has revealed varying degrees of intrinsic disorder in all five viral proteins . While Phosphoprotein (P) shows the maximum level of intrinsic disorder, Glycoprotein G also contains significant intrinsically disordered protein regions (IDPRs) .

Functional Implications of Disorder:

• Conformational Flexibility: Intrinsically disordered regions provide the conformational flexibility necessary for pH-dependent transitions between pre-fusion and post-fusion states .

• Molecular Recognition: These regions often contain disorder-based binding regions, also known as molecular recognition features (MoRFs), which function as potential protein-protein interaction sites .

• Immune Evasion: The structural plasticity provided by disordered regions may contribute to immune evasion by allowing conformational variability in antigenic sites.

• Adaptability: Disorder enables the protein to adapt to different environments encountered during the viral life cycle.

Molecular dynamics simulations extending to 500 ns have demonstrated the flexibility of CHPV proteins and helped characterize their disordered regions . This computational approach complements experimental methods in understanding the conformational dynamics of Glycoprotein G.

The identification of disordered regions and MoRFs in CHPV Glycoprotein G suggests potential targets for disorder-based drug design approaches, offering novel therapeutic strategies against Chandipura virus infection .

What methodologies are most effective for evaluating recombinant Glycoprotein G-based vaccine candidates?

Comprehensive evaluation of recombinant Glycoprotein G-based vaccine candidates requires a multi-faceted approach spanning immunological assessment and protection studies:

Immunization Protocol Optimization:
Research indicates that a standard protocol involves:

• Dose: 1 μg of recombinant Glycoprotein (rGp) per immunization

• Schedule: 3 doses administered 4 weeks apart

• Route: Typically intraperitoneal or intramuscular administration

Humoral Immunity Assessment:

• Antibody Response Kinetics:

  • ELISA to detect anti-CHPV IgG antibodies

  • Monitoring seroconversion timeline (observed as early as 2 weeks post-immunization)

  • Measurement of antibody titers in relation to immunogen concentration

• Neutralization Capacity:

  • In vitro neutralization assays using both homologous and heterologous virus strains

  • Assessment of cross-protective potential (antibodies from immunized mice have demonstrated ability to neutralize heterologous viruses)

Cellular Immunity Evaluation:

• T Cell Response:

  • T cell proliferation assays against rGp (studies have shown approximately 60% T cell proliferation in immunized mice)

  • Cytokine profiling to characterize the T helper cell response

Protection Studies:

• Challenge Model:

  • Intracerebral challenge with 100 LD₅₀ of homologous virus strain

  • Monitoring survival rates (90% protection has been demonstrated in immunized mice)

  • Evaluation of clinical signs and viral load in surviving animals

Data Analysis Framework:

This comprehensive evaluation approach confirms that recombinant Glycoprotein G induces both arms of the immune response (humoral and cellular), representing an ideal vaccine candidate for further development .

How does the structure of Chandipura virus Glycoprotein G compare with other vesiculovirus glycoproteins?

Structural comparison between Chandipura virus Glycoprotein G (CHAV-G) and other vesiculovirus glycoproteins, particularly Vesicular Stomatitis Virus G (VSV-G), reveals important evolutionary insights:

Structural Conservation:

• CHAV-G and VSV-G share high structural similarity in their post-fusion conformations, reflecting their evolutionary relationship

• Both proteins comprise three domains: fusion domain, pleckstrin homology domain (PHD), and trimerization domain

• Both adopt classic hairpin conformations in their post-fusion states

• Both combine features of class I and class II viral fusion proteins in their fusion mechanisms

Regions of Divergence:
The pleckstrin homology domain (PHD) shows the highest divergence among the three domains . This divergence is most pronounced in the major antigenic site, which is located in the most exposed domain of the pre-fusion conformation . The structural differences in this region likely reflect selective pressure from the host immune response, representing a viral adaptation strategy.

Functional Implications:
Despite structural differences, both CHAV-G and VSV-G:

• Trigger fusion at similar pH thresholds (below 6.5)

• Exhibit reversible pH-induced membrane association

• Have pre-fusion trimers that are not stable in solution

Evolutionary Adaptations:
CHAV-G has evolved:

• Alternate structural solutions in hinge regions between PH and fusion domains

• Distinct pH-sensitive switches compared to VSV-G

• Unique antigenic properties that may contribute to its ability to infect humans, whereas VSV primarily infects livestock

This comparative structural analysis provides critical insights for understanding vesiculovirus evolution and host adaptation. The selective pressure on the major antigenic site demonstrates how CHAV has evolved to maintain essential fusion functionality while evading host immune responses, potentially explaining its emergence as a human pathogen .

What are the key protein-protein interactions of Glycoprotein G in the viral life cycle?

Understanding the protein-protein interactions of Chandipura virus Glycoprotein G is essential for elucidating its role in viral assembly, entry, and pathogenesis:

Interactions with Other Viral Proteins:
Experimental studies using ELISA and GST pull-down assays have demonstrated specific interactions between viral proteins:

• G-M Protein Interaction:

  • Glycoprotein G interacts with Matrix (M) protein

  • This interaction is critical for viral assembly and budding

  • Similar interaction patterns have been observed in the related Vesicular Stomatitis Virus

• G-N Protein Interaction:

  • G protein also interacts with Nucleoprotein (N)

  • This interaction may facilitate the incorporation of ribonucleoprotein complexes into virions during assembly

• Absence of G-P Interaction:

  • Interestingly, G protein does not interact with Phosphoprotein (P)

  • This selective interaction pattern parallels that observed in Vesicular Stomatitis Virus

Interaction with Host Cell Receptors:
While specific cellular receptors for CHPV have not been fully characterized, Glycoprotein G is responsible for:

• Recognition of host cell receptors, initiating the infection process

• Binding to cell surface molecules that trigger endocytosis of the virion

pH-Dependent Conformational Changes and Membrane Interactions:
During viral entry:

• Low pH in endosomes (below 6.5) triggers conformational changes in G protein

• These changes expose fusion peptides that insert into endosomal membranes

• G protein forms a hairpin structure that brings viral and cellular membranes together

• This conformational rearrangement drives membrane fusion and release of viral contents

The pH-dependent membrane association of G protein is reversible, an unusual property among viral fusion proteins that may have implications for the fusion mechanism .

Understanding these interactions provides potential targets for antiviral intervention. Disrupting key protein-protein interactions, particularly those involved in viral assembly (G-M) or entry (G-receptor binding), could lead to novel therapeutic approaches against Chandipura virus infection.

What methodological approaches are used to analyze the pH-dependent conformational changes in Glycoprotein G?

Investigating the pH-dependent conformational changes in Chandipura virus Glycoprotein G requires sophisticated methodological approaches spanning structural, biophysical, and computational techniques:

Structural Determination Methods:

• X-ray Crystallography:

  • Determination of high-resolution structures at different pH values

  • The post-fusion conformation of CHAV-G has been crystallized and its structure determined at 3.6Å resolution

  • Challenges include stabilizing the pre-fusion conformation for crystallization

• Cryo-electron Microscopy:

  • Visualization of G protein on viral particles at different pH conditions

  • Allows observation of conformational ensembles rather than single states

Biophysical Characterization Techniques:

• Spectroscopic Methods:

  • Circular dichroism (CD) to monitor secondary structure changes with pH

  • Intrinsic tryptophan fluorescence to detect tertiary structure alterations

  • FTIR spectroscopy to track changes in protein structural elements

• Hydrodynamic Techniques:

  • Size exclusion chromatography to assess oligomeric state changes

  • Analytical ultracentrifugation to determine sedimentation properties at varying pH

  • Dynamic light scattering to measure hydrodynamic radius changes

• Membrane Interaction Studies:

  • Liposome binding assays at different pH values

  • Membrane fusion assays using fluorescently labeled membranes

  • Surface plasmon resonance to quantify binding kinetics to membrane models

Biochemical Approaches:

• Limited Proteolysis:

  • Exposure of different protease-sensitive sites at varying pH

  • Mass spectrometry analysis of digestion patterns

  • Mapping of regions that undergo conformational changes

• Cross-linking Studies:

  • pH-dependent accessibility of residues for cross-linking reagents

  • Identification of residues in proximity in different conformational states

Computational Methods:

• Molecular Dynamics Simulations:

  • All-atom simulations of G protein at different pH conditions

  • Analysis of pH-sensitive switches and conformational transitions

  • Simulation timescales of 500 ns or longer to capture relevant dynamics

• pKa Calculations:

  • Prediction of protonation states of titratable residues at different pH values

  • Identification of pH-sensing residues involved in conformational switching

Functional Correlation Studies:

• Structure-Function Analysis:

  • Mutagenesis of predicted pH-sensitive residues

  • Functional assays to correlate structural changes with fusion activity

  • Analysis of how alterations in pH-sensitive regions affect viral entry

Research has established that CHAV-G undergoes fusion at low pH below 6.5, similar to VSV-G . The protein transitions from its pre-fusion to post-fusion conformation through a series of intermediate states. These conformational changes expose fusion peptides that insert into the endosomal membrane, driving the fusion process necessary for viral entry .

How can molecular dynamics simulations enhance our understanding of Glycoprotein G functionality?

Molecular dynamics (MD) simulations provide unique insights into the dynamic behavior of Chandipura virus Glycoprotein G that are difficult to capture with experimental techniques alone:

Simulation Parameters and Setup:

• System Construction:

  • Atomic models of CHAV-G embedded in appropriate environments (solution, membrane)

  • Explicit water molecules and physiological ion concentrations

  • Application of suitable force fields for proteins, lipids, and solvent

• Simulation Regimes:

  • Extended timescales (up to 500 ns) to observe relevant conformational changes

  • Multiple replicate simulations to improve sampling

  • NPT ensemble (constant particle number, pressure, and temperature)

  • Temperature control at physiological conditions (310K)

Key Applications for CHAV-G Research:

• Intrinsically Disordered Regions Analysis:
  MD simulations have been instrumental in characterizing the flexibility of CHPV proteins, particularly the intrinsically disordered regions . These simulations reveal:

  • Conformational ensembles adopted by disordered segments

  • Transient secondary structure formation

  • Potential molecular recognition features (MoRFs)

  • Dynamic behavior that cannot be captured in static crystal structures

• pH-Dependent Conformational Changes:
  Simulations can model the effects of pH changes by altering protonation states of key residues:

  • Identification of pH-sensitive switches that trigger conformational transitions

  • Characterization of intermediate states during pre-to-post fusion conversion

  • Energetic barriers between conformational states

  • Mechanisms of reversible pH-dependent membrane association

• Fusion Mechanism Investigation:
  MD provides atomic-level details of the fusion process:

  • Conformational dynamics of fusion peptides

  • Membrane interaction and perturbation mechanisms

  • Cooperative motions that drive membrane fusion

  • Water exclusion at membrane contact sites

Analysis Approaches:

• Global Motion Analysis:

  • Principal component analysis to identify major collective motions

  • Free energy landscapes to map conformational space

  • Correlation analysis to identify functionally important coupled motions

• Local Flexibility Assessment:

  • Root-mean-square fluctuation (RMSF) profiles to identify flexible regions

  • Hydrogen bond analysis to track stabilizing interactions

  • Solvent accessibility calculations for key functional regions

Integration with Experimental Data:
The most powerful approach combines MD simulations with experimental data:

• Using crystal structures as starting points for simulations

• Validating simulation results against biophysical measurements

• Generating testable hypotheses for further experimental investigation

The application of MD simulations to CHAV-G has demonstrated the protein's flexibility and helped characterize its intrinsically disordered regions . This computational approach complements structural biology techniques by providing a dynamic view of Glycoprotein G function, essential for understanding its role in viral entry and for developing targeted interventions.