Recombinant Mokola virus Glycoprotein G (G)

What is Mokola virus Glycoprotein G and how does it function in viral infection?

Mokola virus Glycoprotein G (MOKV G) is a type I transmembrane glycoprotein approximately 500 amino acid residues long (503 for MOKV) that mediates two critical steps in viral entry. First, it binds to cellular receptors, triggering virion endocytosis. Subsequently, in the acidic endosomal environment, G undergoes a conformational change from its pre-fusion to post-fusion state, catalyzing the merger of viral and endosomal membranes to release the viral nucleocapsid into the cytoplasm . This glycoprotein is anchored in the viral membrane by a single α-helical transmembrane segment, with the bulk of its mass located as an N-terminal ectodomain outside the viral membrane .

How does MOKV G structurally and functionally differ from rabies virus glycoprotein?

Sequence analysis reveals that MOKV G shares only 54.3% global similarity with rabies virus glycoprotein . This divergence particularly affects the antigenic sites involved in B-cell responses, providing a molecular basis for the absence of cross-protection between Mokola and rabies viruses . Despite these differences, both glycoproteins share similar functional mechanisms, including receptor binding and pH-dependent conformational changes that facilitate membrane fusion. The structural differences explain why rabies vaccines do not provide protection against MOKV infection, underscoring the need for specific anti-Mokola vaccines .

What is the significance of studying MOKV G in lyssavirus research?

MOKV G research is significant for several reasons. First, as one of the more divergent members of the lyssavirus genus, understanding MOKV G structure and function provides insights into lyssavirus diversity and evolution . Second, the lack of cross-protection between MOKV and rabies virus highlights the need for specific countermeasures against non-rabies lyssaviruses that could potentially emerge as humans encroach on new habitats . Third, the structural characterization of MOKV G enables rational design of mutagenesis experiments and potential development of broad-spectrum lyssavirus vaccines . Finally, understanding the pH-sensitive molecular switches in MOKV G contributes to fundamental knowledge about viral fusion mechanisms .

What has been determined about the crystal structure of MOKV G?

The crystal structure of soluble MOKV G ectodomain has been determined in its post-fusion conformation . For crystallization purposes, researchers replaced the hydrophobic fusion loops with more hydrophilic sequences . The structure revealed that MOKV G in its post-fusion state forms a monomer similar to the protomer of the trimeric post-fusion state of vesicular stomatitis virus (VSV) G . The structure determination also revealed the precise localization of antigenic sites and provided the molecular basis for the reversibility of the conformational change that occurs during fusion .

What pH-sensitive molecular switches control MOKV G conformational changes?

The crystal structure of MOKV G revealed several acidic residues and two histidines that play the role of pH-sensitive molecular switches during the structural transition . Specifically, the long helix that constitutes the core of the post-fusion trimer contains many acidic residues located at the trimeric interface. Several of these residues, aligned along the helix, point toward the trimer axis . These residues must be protonated (at low pH) for the post-fusion trimer to be stable. At high pH, when they are negatively charged, they destabilize the interface, explaining the reversibility of the conformational change .

How do MOKV G antigenic sites correlate with its structure?

The crystal structure of MOKV G allowed precise localization of lyssavirus glycoprotein antigenic sites . Sequence alignment between MOKV G and RABV G enables the positioning of RABV G antigenic sites on the MOKV G structure. When MOKV G domains are repositioned on VSV G pre-fusion structure, it becomes apparent that the antigenic sites are located in the most exposed part of the molecule in its pre-fusion conformation, making them very accessible to antibodies . This structural insight explains the immunological properties of lyssavirus glycoproteins and provides a foundation for rational vaccine design.

What expression systems have been successfully used for producing recombinant MOKV G?

Recombinant MOKV G has been successfully expressed using the baculovirus expression system in Spodoptera frugiperda (insect) cells . Additionally, the glycoprotein has been produced in Drosophila Schneider 2 (S2) cells for structural studies . For the crystal structure determination, researchers created a soluble MOKV G ectodomain with modified fusion loops . The baculovirus-expressed recombinant protein was produced in substantial amounts at the surface of insect cells. Although less strongly glycosylated than the native viral glycoprotein produced in BHK-21 cells, the recombinant protein retained antigenic and immunological similarity to the native form .

How can researchers design and construct rabies viral vectors for MOKV G expression?

The design and construction of rabies viral vectors for MOKV G expression involve several key steps:

• Select an appropriate plasmid backbone (e.g., pSADΔG-F3) that allows cloning of novel genes into the rabies viral genome .

• Amplify the MOKV G open reading frame (ORF) using PCR with high-fidelity polymerase, incorporating appropriate restriction enzyme sites at both ends .

• Clone the MOKV G ORF into the plasmid vector using either traditional restriction enzyme-based methods or newer technologies like the In-Fusion system, which allows directional cloning without requiring digestion of PCR products .

• Verify the correct insertion and sequence of the MOKV G gene through restriction enzyme analysis and DNA sequencing .

• For recovery of recombinant virus, transfect the constructed plasmid along with helper plasmids encoding rabies virus N, P, and L proteins and T7 RNA polymerase into suitable mammalian cells .

What are the critical factors affecting the yield and quality of recombinant MOKV G?

Several critical factors affect the yield and quality of recombinant MOKV G:

• Expression system selection: Different expression systems (baculovirus, mammalian, etc.) result in varying glycosylation patterns. While insect cell-expressed MOKV G is less strongly glycosylated than mammalian cell-produced protein, it still retains important antigenic and immunological properties .

• Construct design: For structural studies, modification of hydrophobic fusion loops with more hydrophilic sequences improves solubility and crystallization properties .

• Purification strategy: Appropriate purification protocols are essential to maintain the protein's structural integrity and functional properties.

• pH conditions: Given the pH-sensitivity of MOKV G conformational states, maintaining appropriate pH during purification is critical for preserving the desired conformation (pre-fusion or post-fusion) .

• Biosafety considerations: Production must be performed in biosafety level 2 (BSL-2) facilities with proper procedures for decontamination and disposal of waste materials .

How effective is recombinant MOKV G as a vaccine candidate?

Recombinant MOKV G produced in insect cells using the baculovirus expression system has demonstrated significant protective efficacy as a vaccine candidate . Despite being less strongly glycosylated than the native viral glycoprotein, the recombinant protein is antigenically and immunologically similar to the native form. It is recognized by specific monoclonal antibodies and, most importantly, protects mice against an intracerebral challenge with Mokola virus . This recombinant protein constitutes the first experimental genetically engineered vaccine against a rabies-related virus and meets international standards for protection .

What methods can be used to assess the conformational changes of MOKV G?

Several experimental approaches have been employed to study the pH-dependent conformational changes of MOKV G:

• X-ray crystallography: Used to determine the high-resolution structure of MOKV G in its post-fusion conformation .

• Negative staining electron microscopy (EM): Employed to detect post-fusion trimers of soluble MOKV G ectodomain interacting with membranes and full-length MOKV G at the surface of VSV pseudotypes . EM also revealed that, like VSV G, MOKV G in its post-fusion conformation forms regular networks .

• pH-controlled experiments: By exposing MOKV G to different pH conditions, researchers can trigger and study the conformational transitions between pre-fusion and post-fusion states .

• Pseudotyping: MOKV G can be displayed on the surface of VSV particles (pseudotyping), allowing the study of its conformational changes in a viral context .

How does MOKV G interact with cellular membranes during fusion?

During fusion, MOKV G undergoes significant conformational changes that facilitate viral and endosomal membrane merging. In the low pH environment of endosomes, MOKV G transitions from its pre-fusion to post-fusion conformation . This conformational change exposes hydrophobic fusion loops that insert into the target endosomal membrane . Electron microscopy has revealed that post-fusion trimers of soluble MOKV G ectodomain interact with membranes, and full-length MOKV G at the surface of VSV pseudotypes adopts the trimeric post-fusion conformation at low pH . Additionally, MOKV G in its post-fusion conformation has a tendency to reorganize into regular arrays, which may facilitate the membrane fusion process .

What techniques are recommended for studying structure-function relationships in MOKV G?

For advanced structure-function relationship studies of MOKV G, researchers can employ the following techniques:

• Rational mutagenesis: Based on the crystal structure, researchers can perform targeted mutations of specific residues, particularly those involved in pH sensing or forming the trimeric interface .

• Chimeric glycoprotein construction: Creating chimeras between MOKV G and other lyssavirus glycoproteins (e.g., rabies virus G) can help identify domains responsible for specific functions or antigenic properties .

• Domain swapping experiments: Exchanging domains between pathogenic and non-pathogenic strains to identify elements contributing to pathogenicity .

• Cryo-electron microscopy: For visualizing MOKV G in different conformational states at near-atomic resolution without crystallization requirements.

• Pseudotyping assays: Using VSV or other viral platforms pseudotyped with MOKV G to study entry, fusion, and neutralization in a controlled context .

How can researchers create and utilize MOKV G pseudotyped viruses?

Creating and utilizing MOKV G pseudotyped viruses involves the following steps:

• G-deleted rabies virus construction: Generate G-deleted rabies viral vectors following established protocols for recovery from plasmid DNA .

• Amplification of G-deleted virus: Transfect cells with helper plasmids expressing G protein to produce an initial stock, then amplify to high titers .

• Pseudotyping process: Transfect cells expressing MOKV G protein with G-deleted viruses. The viruses will incorporate MOKV G into their envelope during budding .

• Concentration and purification: Use ultracentrifugation or other concentration methods to prepare high-titer virus stocks .

• Functional testing: Assess the entry capacity and fusion activity of pseudotyped viruses using cell-based assays .

For specific targeting, MOKV G can be pseudotyped with EnvA or EnvB envelopes, allowing selective infection of cells expressing the cognate receptors .

What biosafety considerations are important when working with recombinant MOKV G and related constructs?

When working with recombinant MOKV G and related constructs, researchers must adhere to the following biosafety measures: