Recombinant Vaccinia virus Protein J5 (VACWR097)

What is Vaccinia virus Protein J5 (VACWR097) and what is its role in viral infection?

Protein J5 (VACWR097) is a conserved component of the vaccinia virus entry-fusion complex (EFC), which plays a critical role in poxvirus membrane fusion and cell entry. This protein is conserved across all poxviruses, contains a predicted C-terminal transmembrane domain, and functions as an N-terminally truncated paralog of two other EFC proteins . Research indicates that J5 is essential for virus infectivity, as virions lacking J5 show severely reduced ability to enter host cells despite maintaining normal binding capacity. While the virus yield is only reduced approximately 80% with mild J5 repression, the inability to isolate a complete J5 deletion mutant strongly suggests that this protein serves an essential function in the viral life cycle .

How is J5 structurally integrated into the vaccinia virus entry-fusion complex?

J5 protein is structurally incorporated into the vaccinia virus entry-fusion complex (EFC) as one of its 11 identified components. Experimental evidence from affinity purification studies confirms its physical association with the EFC . The protein's C-terminal transmembrane domain anchors it within the viral membrane, positioning it to participate in the fusion mechanism. As an N-terminally truncated paralog of two other EFC proteins, its structural relationship with these related proteins suggests evolutionary conservation of function. The precise arrangement of J5 within the three-dimensional architecture of the EFC remains an active area of investigation, but its physical integration is essential for complex functionality, as J5-deficient virions demonstrate severe defects in core entry and cannot induce syncytium formation .

What experimental systems are most suitable for studying J5 function?

The most effective experimental systems for studying J5 function involve combination approaches that regulate protein expression. Based on recent research, inducible gene expression systems coupled with RNA silencing provide the most stringent control of J5 levels . Specifically, creating recombinant vaccinia viruses with an inducible J5 gene allows controlled expression, while simultaneously targeting J5 mRNA with siRNA achieves more complete repression. This dual approach has demonstrated a 60-fold reduction in virus yield, compared to 4-5 fold with transcriptional repression alone or 2-fold with RNA silencing alone . For imaging studies, fluorescently tagged J5 constructs can be incorporated into recombinant viruses to track localization during entry. Cell lines permissive for vaccinia virus infection (particularly BS-C-1 and HeLa cells) represent standard models for evaluating J5 function in the context of viral entry.

What techniques provide the most reliable results for analyzing J5's interactions with other EFC components?

Affinity purification techniques have proven effective for confirming J5's association with the entry-fusion complex . For more detailed interaction studies, researchers should consider implementing:

• Proximity Labeling Methods: BioID or APEX2 fusion constructs with J5 can identify proteins in close proximity during the dynamic process of membrane fusion.

• Cross-linking Mass Spectrometry (XL-MS): This approach can capture transient interactions between J5 and other EFC components by creating covalent bonds before mass spectrometric analysis.

• Förster Resonance Energy Transfer (FRET): By tagging J5 and potential interaction partners with appropriate fluorophores, researchers can assess protein-protein interactions in real-time during viral entry.

• Co-immunoprecipitation with Staged Viral Entry: Performing co-IP experiments at different time points during viral entry provides temporal resolution of J5's changing interaction partners.

When designing these experiments, it's critical to consider that the transmembrane domain of J5 presents challenges for maintaining protein solubility and native conformation. Detergent selection is crucial, with mild non-ionic detergents like digitonin or DDM often providing the best balance between solubilization and preservation of protein-protein interactions .

How does the combination of transcriptional repression and RNA silencing enhance investigation of J5 function?

The combination of transcriptional repression and RNA silencing represents a powerful methodological approach for studying essential viral proteins like J5. This dual-targeting strategy operates through distinct mechanisms to achieve more complete protein depletion :

This synergistic effect occurs because any leaky transcription that escapes repression can be targeted by the RNA silencing machinery. The approach has revealed that J5 is essential for core entry and syncytium formation, functions that would have been difficult to discern with less stringent knockdown methods . For researchers studying other essential viral proteins, this methodological combination provides a valuable alternative to deletion mutants when such mutants cannot be isolated. The technique is particularly valuable for temporal studies, as it allows observation of the immediate effects of protein depletion before secondary consequences arise.

What are the key challenges and solutions in generating J5 expression constructs?

Generating functional J5 expression constructs presents several technical challenges:

• Transmembrane Domain Interference: The C-terminal transmembrane domain can cause protein aggregation when expressed in heterologous systems.

  • Solution: Design constructs with removable purification tags positioned to avoid disrupting membrane insertion. Consider using mammalian expression systems rather than bacterial systems.

• Maintaining Native Conformation: Ensuring the recombinant protein adopts its natural structure.

  • Solution: Express J5 in eukaryotic cells such as insect cells (baculovirus system) or mammalian cells that provide appropriate post-translational modifications and membrane environments.

• Low Expression Levels: As an essential viral protein, high-level expression may be toxic.

  • Solution: Use tightly regulated inducible expression systems such as Tet-On systems with optimized induction conditions .

• Purification Challenges: Membrane proteins require specialized purification approaches.

  • Solution: Employ gentle solubilization with detergents like digitonin or DDM, followed by affinity chromatography and size exclusion chromatography in detergent micelles or nanodiscs.

When designing experiments with J5 constructs, researchers should validate protein functionality through complementation assays, where the construct is tested for its ability to rescue infectivity in J5-deficient or J5-repressed viruses .

What specific evidence indicates J5's role in membrane fusion during viral entry?

Several experimental findings provide compelling evidence for J5's critical role in membrane fusion during viral entry:

• Syncytium Formation Defects: Virions with depleted J5 protein demonstrated a complete inability to induce syncytium formation, even under low-pH conditions that normally trigger membrane fusion . This directly links J5 to the fusion machinery.

• Core Entry Inhibition: J5-deficient virions show specific defects in core entry despite maintaining normal binding to host cells. This indicates that J5 functions downstream of attachment, precisely at the fusion step of entry .

• Association with Known Fusion Proteins: Affinity purification studies have confirmed that J5 physically associates with the entry-fusion complex (EFC), which contains other proteins with established roles in membrane fusion .

• Structural Characteristics: The predicted C-terminal transmembrane domain of J5 is consistent with proteins involved in membrane destabilization and fusion processes .

• Evolutionary Conservation: J5 is conserved across all poxviruses and shares structural features with other fusion-related proteins, suggesting functional importance in the conserved entry mechanism .

These findings collectively establish J5 as an integral component of the poxvirus fusion machinery, potentially contributing to the conformational changes that drive membrane merger during viral entry.

How do J5-deficient virions compare to wild-type virions in structural and functional analyses?

J5-deficient virions exhibit specific defects while maintaining other viral properties intact, providing insight into J5's precise function:

These comparative analyses reveal that J5 functions specifically in the membrane fusion and core entry steps of infection, rather than in virion assembly or initial attachment. The fact that J5-deficient virions maintain normal structure but lose fusion capability highlights J5's specialized role in the entry mechanism rather than a structural contribution to virion integrity .

What molecular features of J5 are considered critical for its function in the entry-fusion complex?

Several molecular features of the J5 protein appear to be critical for its function in the entry-fusion complex:

• C-terminal Transmembrane Domain: The predicted transmembrane region anchors J5 within the viral membrane and likely positions it correctly for participation in fusion events . This domain may directly contribute to membrane destabilization during the fusion process.

• Paralogous Relationship: As an N-terminally truncated paralog of two other EFC proteins, specific conserved regions shared between these proteins likely contribute to a common functional mechanism in the fusion process .

• Protein-Protein Interaction Motifs: Although not explicitly detailed in the search results, J5 must contain interaction surfaces that mediate its association with other EFC components, as confirmed by affinity purification studies .

• Conservation Across Poxviruses: The universal conservation of J5 across all poxviruses suggests that its sequence contains essential elements that cannot be substantially altered without compromising function .

The precise structural elements and amino acid residues that are indispensable for J5 function remain an active area of investigation. Systematic mutagenesis studies focusing on conserved residues, particularly those shared with its paralogous proteins, would likely yield valuable insights into the specific molecular features that drive J5's contribution to viral entry.

How can J5 research contribute to the development of antiviral strategies against poxviruses?

Research on the J5 protein offers several promising avenues for antiviral development against poxviruses:

• Entry Inhibitor Design: As an essential component of the entry-fusion complex, J5 represents an attractive target for small molecule inhibitors that could block viral entry. The inability to isolate J5 deletion mutants underscores its critical nature and suggests that viral resistance to J5-targeting drugs might be difficult to develop .

• Broad-Spectrum Potential: The conservation of J5 across all poxviruses indicates that interventions targeting this protein could potentially be effective against multiple poxvirus pathogens, including emerging threats .

• Rational Vaccine Design: Understanding J5's role in entry could inform the development of recombinant vaccinia virus vaccines with modified J5 that retains immunogenicity while reducing pathogenicity. This builds on the established platform of recombinant vaccinia viruses for vaccination against other infectious agents .

• Combination Approaches: The synergistic effects observed when combining transcriptional repression and RNA silencing of J5 suggest that combination therapies targeting viral entry through multiple mechanisms might be particularly effective .

• Screening Platforms: Developing high-throughput screening systems using J5-dependent entry as a readout could facilitate the discovery of novel antiviral compounds that specifically disrupt the function of this essential protein.

Current challenges include developing delivery methods for potential therapeutics and ensuring specificity to avoid disrupting host membrane fusion processes. Future research directions should include structural studies of J5 to facilitate structure-based drug design approaches.

What are the most significant knowledge gaps regarding J5 protein that require further investigation?

Despite progress in understanding J5's importance in vaccinia virus entry, several critical knowledge gaps remain:

• Structural Information: No high-resolution structural data for J5 is currently available. Determining the three-dimensional structure through X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy would significantly advance understanding of its function and facilitate structure-based drug design.

• Mechanistic Details: The precise molecular mechanism by which J5 contributes to membrane fusion remains unclear. Does it directly participate in membrane destabilization, or does it regulate the activity of other fusion proteins? Time-resolved studies of the fusion process are needed.

• Host Protein Interactions: Potential interactions between J5 and host cell proteins during entry have not been thoroughly characterized. Identifying such interactions could reveal novel antiviral targets and improve understanding of poxvirus tropism.

• Dynamic Conformational Changes: How J5 might undergo conformational changes during the fusion process is unknown. Studies employing techniques like hydrogen-deuterium exchange mass spectrometry or single-molecule FRET could illuminate these dynamics.

• Functional Domains: The specific functional domains within J5, particularly those that distinguish it from its paralogous proteins, require mapping through systematic mutagenesis studies .

• Integration with Other EFC Components: The spatial and functional relationships between J5 and the other 10 EFC proteins during the coordinated process of membrane fusion need clarification .

Addressing these knowledge gaps will require innovative approaches combining structural biology, molecular virology, and advanced imaging techniques to build a comprehensive understanding of this essential viral protein.

What methodological approaches can be used to study the evolution and conservation of J5 across different poxviruses?

To study the evolution and conservation of J5 across poxviruses, researchers can employ several complementary methodological approaches:

• Comparative Genomics and Phylogenetics:

  • Sequence alignment of J5 homologs from diverse poxvirus species to identify conserved motifs and variable regions

  • Phylogenetic analysis to reconstruct the evolutionary history of J5 relative to virus speciation events

  • Calculation of selection pressures (dN/dS ratios) to identify regions under purifying or positive selection

• Structural Bioinformatics:

  • Protein structure prediction using AlphaFold2 or similar tools to compare predicted structures across poxvirus species

  • Mapping of conserved residues onto structural models to identify functionally important surfaces

  • Analysis of co-evolution between J5 and other EFC components across poxvirus species

• Functional Complementation Studies:

  • Construction of chimeric J5 proteins incorporating regions from different poxviruses

  • Testing the ability of J5 from diverse poxviruses to complement a J5-deficient vaccinia virus

  • Systematic mutagenesis of conserved residues to determine their functional importance

• Experimental Evolution:

  • Directed evolution experiments applying selective pressure on J5 function

  • Deep sequencing to track mutations that arise in J5 during serial passage under various conditions

  • Characterization of compensatory mutations in other EFC components that co-evolve with J5 variants

These approaches would provide insights into which aspects of J5 structure and function have been conserved throughout poxvirus evolution, potentially revealing the protein's core mechanistic contributions to viral entry. Additionally, identifying variability in J5 across host-specific poxviruses might illuminate how this protein contributes to viral tropism and host range determination.

What are the optimal conditions for expressing and purifying recombinant J5 protein while maintaining its native conformation?

Expressing and purifying membrane proteins like J5 with retained native conformation requires careful optimization:

• Expression Systems:

  • Mammalian Cell Expression: HEK293 or CHO cells provide appropriate post-translational modifications and membrane environments for J5 expression

  • Insect Cell Expression: Baculovirus systems in Sf9 or High Five cells offer high yields while maintaining eukaryotic processing

  • Cell-Free Expression: Recent advances in cell-free systems with nanodiscs or liposomes can directly incorporate J5 into membrane mimetics

• Expression Construct Design:

  • Include affinity tags (His6, FLAG, or Strep-tag II) positioned to avoid interference with the C-terminal transmembrane domain

  • Consider fusion partners (MBP, SUMO) to enhance solubility, with cleavable linkers

  • Use strong but controllable promoters (CMV for mammalian cells, polyhedrin for baculovirus)

• Membrane Extraction:

  • Gentle solubilization with digitonin (0.5-1%) or DDM (0.5-1%) preserves protein-protein interactions

  • Extraction at 4°C for 1-2 hours with constant gentle rotation

  • Avoid harsh detergents like SDS or Triton X-100 that may denature the protein

• Purification Strategy:

  • Two-step purification: affinity chromatography followed by size exclusion chromatography

  • Maintain detergent above critical micelle concentration throughout purification

  • Include glycerol (10%) and reducing agents to stabilize the protein

• Reconstitution Options:

  • Nanodiscs with appropriate phospholipid composition

  • Proteoliposomes for functional studies

  • Amphipols for long-term stability

• Validation of Native Conformation:

  • Circular dichroism to assess secondary structure

  • Limited proteolysis to probe folding

  • Functional complementation assays to confirm activity

When working with J5, researchers should be particularly attentive to protein aggregation and monitor sample homogeneity using dynamic light scattering or analytical ultracentrifugation throughout the purification process.

What approaches are most effective for quantitatively assessing J5's contribution to viral entry?

Several quantitative approaches can effectively assess J5's contribution to viral entry:

• Core Delivery Assays:

  • Viruses expressing fluorescently tagged core proteins can be tracked by confocal microscopy

  • Quantification of core entry events in cells infected with J5-repressed virions versus control virions

  • Image analysis software can provide statistical measures of core delivery efficiency

• Fusion-from-Without (FFWO) Assays:

  • Measurement of syncytium formation induced by viral particles bound to cells under low pH conditions

  • Quantification of multinucleated cells formed by J5-containing versus J5-deficient virions

  • This approach specifically assesses the fusion capability independent of other entry steps

• Luciferase Reporter Systems:

  • Recombinant viruses containing luciferase genes under early viral promoters

  • Luminescence measurements provide quantitative readout of successful entry and early gene expression

  • Comparison between J5-normal and J5-deficient virions yields quantitative measures of entry efficiency

• Time-of-Addition Studies:

  • Application of entry inhibitors at different time points to determine the temporal window of J5 action

  • Quantification of infection rates as a function of inhibitor addition time

  • This approach can precisely define when during entry J5 functions

• Single-Virus Tracking:

  • Real-time fluorescence microscopy of individual viral particles during entry

  • Measurement of hemifusion and complete fusion kinetics

  • Comparison between wild-type and J5-deficient virions reveals specific steps affected

The combination of transcriptional repression and RNA silencing provides an excellent experimental platform for these assays, as it achieves a more complete reduction in J5 levels (60-fold reduction in virus yield) than either method alone . For maximal rigor, multiple independent approaches should be combined to build a comprehensive quantitative understanding of J5's role.

How does understanding J5 function contribute to the broader knowledge of poxvirus entry mechanisms?

Understanding J5 function provides several critical insights into the broader mechanisms of poxvirus entry:

• EFC Component Interactions: J5 research has helped establish that the entry-fusion complex functions as an integrated unit requiring all components for efficient entry. The severe defects observed with J5 depletion, despite the presence of all other EFC proteins, demonstrates the non-redundant nature of these components .

• Sequential Steps in Viral Entry: Studies of J5-deficient virions have helped delineate the sequence of events during poxvirus entry. The observation that these virions maintain normal binding but fail at core entry and syncytium formation places J5 function specifically at the fusion stage rather than attachment .

• Model for Membrane Fusion: J5's predicted transmembrane domain and essential role in fusion contribute to an emerging model where multiple viral proteins coordinate to induce membrane curvature, hemifusion, and pore formation during entry.

• Evolutionary Insights: The conservation of J5 across all poxviruses, together with its relationship as an N-terminally truncated paralog of other EFC proteins, suggests evolution through gene duplication and specialization events that shaped the complex poxvirus entry machinery .

• Methodological Advances: The development of combined transcriptional repression and RNA silencing approaches for studying J5 has established a paradigm for investigating other essential viral proteins .

By integrating J5 research findings with studies of other EFC components, researchers can build a comprehensive model of the poxvirus entry mechanism, which differs significantly from the better-understood entry processes of many other enveloped viruses. This distinctive entry mechanism may explain the broad cell tropism characteristic of poxviruses.

What experimental approaches can determine whether J5's function is conserved in recombinant vaccinia viruses expressing foreign antigens?

To determine whether J5's function is conserved in recombinant vaccinia viruses expressing foreign antigens, researchers can implement several experimental approaches:

• Comparative Entry Kinetics:

  • Measure entry rates of wild-type vaccinia virus versus recombinant virus expressing foreign antigens

  • Use fluorescently labeled virions and live-cell imaging to track entry events in real-time

  • Quantify any differences in fusion efficiency or core delivery timing

• J5 Depletion in Recombinant Viruses:

  • Apply the dual transcriptional repression and RNA silencing approach to J5 in recombinant viruses

  • Compare the impact on infectivity between wild-type and recombinant viruses

  • Determine whether foreign antigen expression alters the dependency on J5 for entry

• J5 Protein Interaction Analysis:

  • Conduct affinity purification of J5 from both wild-type and recombinant viruses

  • Compare the composition of co-purifying proteins using mass spectrometry

  • Assess whether foreign antigens disrupt normal J5 interactions within the EFC

• Structural Analysis of Viral Particles:

  • Utilize cryo-electron microscopy to examine the organization of envelope proteins in recombinant viruses

  • Compare the localization and distribution of J5 in wild-type versus recombinant virions

  • Determine if foreign antigen expression alters membrane curvature or EFC arrangement

• Syncytium Formation Assays:

  • Compare the ability of wild-type and recombinant viruses to induce cell-cell fusion under low pH conditions

  • Quantify any differences in fusion efficiency that might indicate altered J5 function

These approaches are particularly relevant because recombinant vaccinia viruses are valuable tools for vaccine development and gene therapy . Understanding whether foreign antigen expression impacts J5 function and viral entry efficiency would provide important insights for optimizing these applications.

How can knowledge about J5 inform the design of safer recombinant vaccinia virus vectors for vaccine and therapeutic applications?

Knowledge about J5 can significantly contribute to designing safer recombinant vaccinia virus vectors through several strategies:

• Conditional Modulation of J5 Expression:

  • Engineering vectors with inducible or tissue-specific J5 expression could restrict viral replication to target tissues

  • This approach would maintain immunogenicity while limiting undesired viral spread

• Attenuated J5 Variants:

  • Identifying mutations in J5 that reduce fusion efficiency without eliminating it entirely

  • These partial loss-of-function variants could generate attenuated vectors with improved safety profiles

  • The dual transcriptional repression and RNA silencing approach provides a platform for testing such variants

• Host Range Modification:

  • Understanding how J5 contributes to host range could allow engineering of vectors with restricted tissue tropism

  • This would be particularly valuable for oncolytic applications where viral replication should be limited to tumor cells

• Integration with Existing Attenuated Strains:

  • Combining optimized J5 variants with already attenuated vaccinia strains like MVA or NYVAC

  • These strains already have multiple gene deletions, and further J5 modification could enhance their safety

• Polyvalent Vaccine Design:

  • Knowledge of J5's position and function in the viral membrane can inform optimal placement of foreign antigens

  • Strategic positioning relative to J5 and the EFC could improve antigen presentation while maintaining vector safety

Implementation of these approaches would build upon the established advantages of vaccinia virus vectors, including heat stability, low cost, ease of administration, and the ability to stimulate both humoral and cell-mediated immunity . By targeting J5 function, researchers can develop vectors that maintain these benefits while addressing safety concerns, particularly for immunocompromised individuals or other vulnerable populations.