Recombinant Dhori virus Envelope glycoprotein (P4)

What is Dhori virus and what is the structural classification of its envelope glycoprotein?

Structural studies have determined both pre-fusion and post-fusion conformations of the Dhori virus envelope glycoprotein using cryo-electron microscopy at a resolution of 3.3 Å . The structural analyses confirmed that despite sharing only approximately 28% sequence identity with baculovirus Gp64s, Dhori virus envelope glycoprotein shows remarkable structural homology to these insect virus proteins . This similarity supports the hypothesis of a common evolutionary ancestor between thogotoviruses and baculoviruses, despite their different host ranges.

How is the Dhori virus envelope glycoprotein organized in terms of domains and functional regions?

The Dhori virus envelope glycoprotein contains distinct domains with varying degrees of conservation and functionality:

The fusion loops within Domain I are particularly important as they contain hydrophobic amino acids that are conserved between Dhori thogotovirus GP, QRFV_Hyp, and baculovirus GP64. This conservation enables these proteins to insert into target membranes and induce membrane fusion . The variability observed in Domain II is hypothesized to be related to the distinct host tropism exhibited by different thogotoviruses .

What is known about the molecular weight and post-translational modifications of Dhori virus envelope glycoprotein?

The envelope glycoprotein (GP) of Dhori virus undergoes significant post-translational modifications, primarily glycosylation. While the protein has a predicted molecular weight of 58 kDa based on its amino acid sequence, studies have shown that the mature glycoprotein has an observed molecular weight of approximately 75 kDa due to extensive glycosylation . This substantial difference highlights the importance of post-translational modifications in the maturation and functionality of viral envelope proteins.

The glycosylation profile is critical for proper folding, transport, and function of the envelope glycoprotein. It may also play roles in immune evasion and receptor recognition. When analyzing recombinant Dhori virus envelope glycoprotein in laboratory settings, researchers should account for these glycosylation patterns, especially when using expression systems that might alter the glycosylation profile compared to native virus.

What expression systems are optimal for producing recombinant Dhori virus envelope glycoprotein?

Successful production of functional recombinant Dhori virus envelope glycoprotein requires careful consideration of expression systems that can properly process the protein, particularly with regard to glycosylation patterns. Based on research findings:

Research has demonstrated that mammalian cell expression systems are most suitable for producing functional recombinant envelope glycoproteins that retain native-like properties, which is critical for studies involving cell tropism and virus-host interactions . When using recombinant Dhori virus envelope glycoprotein for pseudotyping applications, expression in mammalian cells has shown the most robust results in terms of functional incorporation into lentiviral or other viral vectors .

How can pseudotyping assays be optimized using recombinant Dhori virus envelope glycoprotein?

Pseudotyping viruses with Dhori virus envelope glycoprotein offers valuable research applications for studying viral entry mechanisms and cell tropism without the biosafety concerns associated with infectious thogotoviruses. Research findings indicate several optimization strategies:

• Vector Selection: Lentivirus vectors have demonstrated robust pseudotyping compatibility with Dhori virus envelope glycoprotein, while influenza virus vectors show compatibility but with lower efficiency .

• pH Considerations: Dhori virus envelope glycoprotein exhibits pH-dependent triggering of membrane fusion, so maintaining appropriate pH conditions during production and storage is critical for retaining functionality .

• Cell Type for Production: HEK293T cells have shown high efficiency for producing pseudotyped particles with functional Dhori virus envelope glycoprotein.

• Purification Methods: Ultracentrifugation through sucrose cushions provides optimal purification of pseudotyped particles while maintaining glycoprotein integrity.

The successful generation of replication-competent vesicular stomatitis virus expressing DHOV glycoproteins demonstrates the versatility of this approach . These pseudotyped systems can be used for high-throughput screening of antiviral compounds, studying receptor usage, and examining cell tropism without requiring high-containment facilities.

What are the key considerations for designing fusion assays with recombinant Dhori virus envelope glycoprotein?

Cell-cell fusion assays have proven valuable for studying the fusion mechanism of thogotovirus envelope glycoproteins. When designing such assays with recombinant Dhori virus envelope glycoprotein, researchers should consider:

• pH Triggering: Dhori virus envelope glycoprotein exhibits pH-dependent fusion activation. Research shows that optimal fusion occurs at pH values between 5.0-5.5, mimicking the endosomal environment during viral entry .

• Temperature Conditions: Fusion activity shows temperature dependence, with optimal activity typically observed at 37°C.

• Expression Level Monitoring: Consistent expression levels must be maintained across experimental conditions, as variation can significantly impact fusion efficiency measurements.

• Quantification Methods: Both microscopic analysis of syncytia formation and reporter-based quantitative assays (such as luciferase reporter systems) have been successfully employed to measure fusion activity.

In designing these assays, it's important to include appropriate controls, such as using fusion-defective mutants (particularly those with alterations in the fusion loop region) to validate the specificity of observed fusion events .

What structural features of the Dhori virus envelope glycoprotein are critical for its fusion activity?

The fusion mechanism of Dhori virus envelope glycoprotein involves specific structural elements that undergo conformational changes during the fusion process. Key structural features include:

• Fusion Loops: The fusion domain contains highly conserved hydrophobic amino acids that insert into target membranes to initiate fusion. These residues are conserved across Dhori thogotovirus GP, quaranfil virus hypothetical protein, and baculovirus GP64 .

• Domain Architecture: The post-fusion structure determined for Dhori virus envelope glycoprotein confirms its classification as a class III viral fusion protein, sharing structural homology with baculovirus GP64 despite low sequence identity of approximately 28% .

• pH Sensor Residues: Specific histidine residues likely serve as pH sensors that trigger conformational changes in acidic environments, similar to other class III viral fusion proteins.

• Prefusion Stability Elements: The prefusion conformation is metastable and maintained by specific interactions that are disrupted upon pH-triggered activation .

This structural organization allows the protein to remain in a metastable prefusion state until triggered by the acidic environment of the endosome, whereupon it undergoes dramatic conformational changes that drive membrane fusion .

What does the evolutionary relationship between thogotovirus and baculovirus glycoproteins reveal about viral adaptation?

Structural and phylogenetic analyses of Dhori virus envelope glycoprotein provide compelling evidence for evolutionary connections between thogotoviruses and baculoviruses:

• Structural Conservation: Despite the relatively low sequence identity of approximately 28%, the structural similarity between thogotovirus envelope glycoproteins and baculovirus Gp64 suggests a common evolutionary origin .

• Domain-Specific Evolution: Different domains show varying degrees of conservation. Domain I, containing the fusion loops, is the most conserved region, indicating strong selective pressure to maintain fusion functionality .

• Divergent Adaptation: Domain II shows the highest variability among different viruses, which correlates with adaptation to different host ranges and cell tropism .

• Fusion Mechanism Conservation: The conservation of the fusion loops with their characteristic hydrophobic residues demonstrates the fundamental importance of this mechanism across diverse viral families .

These findings suggest that despite adaptations to different hosts (insects for baculoviruses, mammals including humans for thogotoviruses), the essential fusion machinery has been conserved throughout evolution. This reflects the effectiveness of this particular entry mechanism and suggests that certain structural solutions for membrane fusion have been highly conserved due to their efficiency .

How does the broad tropism of Dhori virus relate to the properties of its envelope glycoprotein?

Research has revealed that Dhori virus exhibits remarkably broad cell tropism, capable of infecting a wide range of mammalian cells, including human primary cells. This promiscuous cell entry behavior is directly linked to properties of its envelope glycoprotein:

• Receptor Ubiquity: The ability of Dhori virus to enter numerous mammalian cell types suggests that its envelope glycoprotein recognizes a widely distributed receptor or utilizes multiple receptors .

• Fusion Mechanism: The pH-dependent fusion mechanism allows the virus to utilize the ubiquitous endocytic pathway for entry, reducing dependence on specific cell surface factors .

• Conserved Entry Pathway: Pseudotyping studies have demonstrated that the envelope glycoprotein alone is sufficient to confer broad tropism to lentiviral and VSV vectors, confirming its central role in determining cell susceptibility .

This broad tropism has significant implications for the zoonotic potential of thogotoviruses, as the ability to infect diverse cell types facilitates cross-species transmission . Furthermore, understanding this broad tropism is essential for risk assessment and surveillance strategies for these emerging pathogens.

What is known about the immunological response to Dhori virus envelope glycoprotein?

Studies of immune responses to thogotoviruses have provided valuable insights into how the host immune system recognizes and responds to the envelope glycoprotein:

• Antibody Targets: Antibody responses in infected animals are primarily directed against the envelope glycoprotein (GP, 58 kDa native/75 kDa glycosylated) and nucleoprotein (NP, 52 kDa), with NP typically eliciting the strongest signals in Western blotting analyses .

• Cross-Reactivity Patterns: Antisera from mice infected with closely related virus isolates show high cross-reactivity within virus clusters (THOV-like or DHOV-like) but limited cross-reactivity between the two groups .

• Neutralizing Epitopes: The envelope glycoprotein contains the primary neutralizing epitopes, making it a key target for protective immunity.

• Glycosylation Effects: The extensive glycosylation of the envelope glycoprotein (increasing apparent molecular weight from 58 kDa to 75 kDa) may impact immunogenicity and antibody recognition .

This immunological profile has important implications for diagnostics, vaccine development, and understanding protection against thogotovirus infections. The limited cross-reactivity between THOV-like and DHOV-like clusters suggests that vaccines may need to incorporate antigens from multiple viral strains to provide broad protection .

How can structural insights into Dhori virus envelope glycoprotein inform antiviral drug design?

The detailed structural characterization of Dhori virus envelope glycoprotein opens several promising avenues for antiviral development:

• Fusion Inhibition: The identified fusion loops represent highly conserved regions that could be targeted by small-molecule inhibitors or peptide-based fusion inhibitors to block the critical membrane fusion step .

• Conformational Locks: Compounds that stabilize the prefusion conformation could prevent the pH-triggered conformational changes required for fusion, effectively neutralizing the virus .

• Broad-Spectrum Potential: The structural similarities between thogotovirus and baculovirus fusion proteins suggest that antivirals targeting conserved features might exhibit activity against multiple viral families .

• Rational Design Approaches: The availability of both pre-fusion and post-fusion structures enables structure-based drug design approaches, including virtual screening and fragment-based drug discovery .

The conservation of key functional elements across the thogotovirus genus suggests that successful inhibitors might demonstrate broad activity against multiple members of this virus family, potentially providing protection against both known and emerging thogotoviruses .

What are the current challenges and recent advances in developing recombinant Dhori virus envelope glycoprotein as a research tool?

While recombinant Dhori virus envelope glycoprotein offers valuable research applications, several challenges and recent advances should be considered:

Recent advances in pseudotyping tools have been particularly significant, with successful development of lentivirus, influenza virus, and VSV-based vectors incorporating functional Dhori virus envelope glycoprotein . These systems enable safe study of viral entry in diverse cell types without requiring work with infectious thogotoviruses.

Additionally, the development of cell-cell fusion assays has provided valuable tools for studying the fusion mechanism and screening potential inhibitors . These methodological advances have expanded the research toolbox available for thogotovirus studies and facilitated more detailed investigations of viral entry mechanisms.