Recombinant Vaccinia virus Protein O1 (VACWR068)

What is the Vaccinia virus O1 protein and what is its function?

The Vaccinia virus O1 protein (VACWR068) is a highly conserved orthopoxvirus protein with a predicted molecular size of approximately 78 kDa. It functions as a positive regulator of the ERK1/2 pathway downstream of the epidermal growth factor receptor (EGFR), complementing the autocrine function of vaccinia virus growth factor (VGF) . The O1 protein is required for sustained activation of the Raf/MEK/ERK pathway in infected cells, which has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture . It enhances virulence and spread of replication-competent vaccinia virus, as demonstrated through studies with deletion mutants .

How is the O1L gene organized in the vaccinia virus genome?

The O1L gene is highly conserved among orthopoxviruses and encodes the O1 protein. In modified vaccinia virus Ankara (MVA), the O1L open reading frame (ORF) is fragmented, whereas in chorioallantois vaccinia virus Ankara (CVA) and other replication-competent vaccinia viruses, it remains intact . The gene is present in both Vaccinia virus and Variola virus (the causative agent of smallpox) . The complete functional O1L gene was successfully reinserted into MVA through genetic engineering, demonstrating that the gene's integrity can be restored experimentally .

What methods are commonly used to purify recombinant O1 protein?

Recombinant Vaccinia virus Protein O1 can be produced in various expression systems including cell-free expression, E. coli, yeast, baculovirus, or mammalian cell systems . After expression, the protein is typically purified to ≥85% purity as determined by SDS-PAGE . Common purification methods include affinity chromatography (using tags such as His-tag or GST-tag), followed by size exclusion chromatography to achieve high purity. For functional studies, it's crucial to ensure that the purified protein maintains its native conformation and activity, which can be verified through specific binding assays or biological activity tests measuring ERK1/2 pathway activation in cell culture systems .

What experimental models are suitable for studying O1 protein function?

Several experimental models have proven valuable for studying O1 protein function:

• Cell Culture Systems: Human 293 cells and other mammalian cell lines have been used to study ERK1/2 activation mediated by O1 protein .

• Viral Genetic Systems: Comparison studies between CVA (with intact O1L gene) and MVA (with fragmented O1L gene) provide insights into O1 protein function .

• Gene Deletion Mutants: CVA-ΔO1L mutants have been generated to study the specific effects of O1 protein absence .

• Gene Reintroduction Systems: Reintroduction of intact O1L gene into MVA has been used to restore function .

• In vivo Mouse Models: BALB/c mice have been utilized to assess virulence and viral spread from lungs to ovaries following intranasal infection .

These models collectively enable comprehensive analysis of O1 protein's role in cellular signaling and viral pathogenesis.

How does the O1 protein interact with the Raf/MEK/ERK signaling pathway at the molecular level?

The O1 protein functions as a positive regulator of the ERK1/2 pathway downstream of the EGFR, complementing the function of VGF by sustaining ERK1/2 activation during the course of infection . While VGF initiates ERK1/2 activation through its interaction with EGFR as an EGF-like protein, O1 protein appears to maintain this activation through a different mechanism .

The precise molecular interactions between O1 protein and components of the Raf/MEK/ERK cascade have not been fully elucidated, but experimental evidence suggests that O1 protein acts at a point in the pathway that is beyond the initial receptor activation but before the final ERK1/2 phosphorylation events . This is supported by the observation that deletion of the O1L gene in CVA caused only transient ERK1/2 activation after infection, while reintroduction of a functional O1L gene into MVA restored sustained ERK1/2 activation .

A comprehensive understanding of these interactions would require protein-protein interaction studies using techniques such as co-immunoprecipitation, yeast two-hybrid screening, or proximity labeling approaches to identify the specific components of the signaling pathway that directly interact with O1 protein.

What are the experimental challenges in differentiating the roles of O1 protein versus VGF in ERK1/2 activation?

Differentiating the roles of O1 protein and VGF in ERK1/2 activation presents several experimental challenges:

• Temporal Dynamics: Both proteins contribute to ERK1/2 activation but potentially at different phases of infection. VGF appears to initiate activation, while O1 sustains it . Time-course experiments with high temporal resolution are necessary to distinguish these effects.

• Pathway Redundancy: The Raf/MEK/ERK pathway can be activated through multiple inputs, creating background noise in experimental systems.

• Knockout Compensation: Single gene knockout studies may be complicated by compensatory mechanisms.

• Protein-Specific Inhibitors: Development of specific inhibitors for each protein would facilitate functional studies but remains technically challenging.

To address these challenges, researchers might employ:

• Double knockout studies (ΔO1L/ΔVGF) compared to single knockouts

• Inducible expression systems allowing temporal control of protein expression

• Pharmacological inhibitors targeting different levels of the signaling pathway to pinpoint where O1 and VGF exert their effects

• Phosphoproteomics to globally analyze pathway activation patterns in the presence and absence of each protein

• CRISPR interference approaches for temporal regulation of gene expression

What structural features of the O1 protein are critical for its function in ERK1/2 pathway modulation?

While the complete three-dimensional structure of the O1 protein has not been fully characterized, understanding its structural features is crucial for elucidating its function. The O1 protein is predicted to be a 78-kDa protein , but the specific domains and motifs responsible for its interaction with the ERK1/2 pathway remain to be determined.

To identify critical structural features of O1 protein, researchers could employ:

• Structural Prediction Tools: Computational approaches such as those described in the CoVES (Combinatorial Variant Effects from Structure) methodology could predict amino acid preferences based on local structural contexts .

• Deletion and Point Mutation Studies: Systematic mutation of conserved regions followed by functional assays for ERK1/2 activation.

• Protein Domain Mapping: Expression of truncated versions of the protein to identify minimal functional domains.

• Conservation Analysis: Comparison of O1 protein sequences across different orthopoxviruses to identify highly conserved regions likely to be functionally important.

• Structural Biology Approaches: X-ray crystallography, cryo-EM, or NMR spectroscopy to determine the three-dimensional structure of the protein or its domains.

The CoVES approach suggests that in many cases, amino acid preferences at individual residues can explain much of the combinatorial mutation effects (R² ~78-98%), indicating that local structural contexts around residues might be sufficient to predict mutation preferences . This approach could be valuable for identifying critical residues in the O1 protein structure.

How can researchers design experiments to determine if O1 protein functions differently in various cell types?

To investigate potential cell type-specific functions of O1 protein, researchers should design comprehensive experimental protocols:

Experimental Design Framework:

• Cell Panel Selection:

  • Choose diverse cell types representing different tissues and species

  • Include permissive and non-permissive cells for vaccinia virus replication

  • Consider primary cells versus immortalized lines

• Infection Protocol:

  • Use wild-type virus and O1L deletion mutants at consistent MOI (multiplicity of infection)

  • Include both CVA (with intact O1L) and MVA (with fragmented O1L) for comparison

  • Establish time-course sampling (0, 2, 4, 8, 12, 24 hours post-infection)

• Analytical Approaches:

  • Quantify ERK1/2 phosphorylation by Western blotting and phospho-specific flow cytometry

  • Measure virus replication kinetics through plaque assays and qPCR

  • Assess cytopathic effects through microscopy and cell viability assays

  • Perform transcriptomics to identify cell-type specific responses

• Validation Methods:

  • Pharmacological inhibition of ERK1/2 pathway components

  • siRNA knockdown of potential host interaction partners

  • Rescue experiments with recombinant O1 protein expression

This experimental framework would allow researchers to determine whether O1 protein functions through the same mechanisms across different cell types or if it exhibits context-dependent activities that might contribute to tissue tropism or host range.

What are the most effective genetic engineering strategies for studying O1 protein function?

Several genetic engineering strategies have proven effective for studying O1 protein function:

• Gene Deletion and Reinsertion: Complete deletion of the O1L gene from replication-competent vaccinia viruses (like CVA) and subsequent reinsertion of the gene provides direct evidence of protein function . This approach has revealed that O1 protein is required for sustained ERK1/2 activation and enhances virulence and viral spread in mice .

• BAC Mutagenesis: Bacterial artificial chromosome (BAC) technology enables precise genetic manipulation of large viral genomes. This was used to generate MVA-BAC+O1L, where the fragmented O1L ORF in MVA was replaced with the intact version from CVA .

• Traceless Gene Deletion: Methods for creating marker-free ("traceless") deletions are particularly valuable as they eliminate potential confounding effects from selection markers . This approach confirmed that the phenotypic effects observed with O1L deletion were due to the absence of the gene itself, not the presence of selection markers .

• Point Mutations and Domain Swapping: Beyond simple deletion/insertion, introducing specific mutations or swapping functional domains between related proteins can provide insights into structure-function relationships.

• Inducible Expression Systems: Tetracycline-regulated or other inducible promoters allow temporal control of O1 protein expression, enabling studies of its role at different stages of infection.

When designing genetic engineering strategies, it's crucial to verify the introduced changes through whole-genome sequencing to confirm no additional mutations have occurred, as demonstrated in the verification of MVA-BAC+O1L .

What are the recommended procedures for generating O1L gene deletion mutants?

Based on successful previous research, the following procedure is recommended for generating O1L gene deletion mutants:

• Selection of Parental Virus: Begin with a well-characterized vaccinia virus strain such as CVA that contains an intact O1L gene .

• Construction of Transfer Plasmid:

  • Design a transfer plasmid containing selection markers (such as rpsL/neo counterselection cassette) flanked by 500-1000 bp homologous sequences from regions upstream and downstream of the O1L gene .

  • For traceless deletion, a two-step process is required where the selection marker is subsequently removed through a second recombination step .

• Recombination Procedure:

  • Infect permissive cells (e.g., CEF or BHK-21) with parental virus.

  • Transfect infected cells with the transfer plasmid.

  • Allow homologous recombination to occur during viral replication.

• Selection of Recombinants:

  • Apply appropriate selection (e.g., G418 for neo resistance) to enrich for recombinant viruses.

  • Plate dilutions to isolate individual plaques.

  • Screen plaques by PCR to identify recombinants.

• Verification of Deletion:

  • Confirm deletion by PCR, restriction enzyme analysis, and sequencing of the target region.

  • For complete confidence, whole-genome sequencing is recommended to ensure no additional mutations occurred .

• Functional Validation:

  • Analyze growth characteristics, plaque morphology, and ERK1/2 activation patterns to confirm the functional consequences of O1L deletion .

This procedure has been successfully implemented to create CVA-ΔO1L variants, demonstrating reduced plaque size, attenuated cytopathic effect, and decreased virulence in mice .

What methods are most reliable for measuring ERK1/2 activation in the context of O1 protein studies?

Several complementary methods provide reliable measurement of ERK1/2 activation in the context of O1 protein studies:

• Western Blotting:

  • Primary method for detecting phosphorylated ERK1/2 (p-ERK1/2) relative to total ERK1/2

  • Samples should be collected at multiple time points post-infection (e.g., 0, 0.5, 1, 2, 4, 8, 12, 24 hours)

  • Use phospho-specific antibodies targeting Thr202/Tyr204 of ERK1/2

  • Include appropriate controls: positive control (EGF treatment), negative control (MEK inhibitor treatment)

  • Quantify band intensity using densitometry for semi-quantitative analysis

• Flow Cytometry with Phospho-Specific Antibodies:

  • Provides single-cell resolution of ERK1/2 activation

  • Allows simultaneous analysis of viral infection markers

  • Particularly useful for heterogeneous populations or time-course studies

• Immunofluorescence Microscopy:

  • Visualizes subcellular localization of activated ERK1/2

  • Can reveal spatial relationships between viral proteins and p-ERK1/2

  • Useful for examining cell-to-cell variability within infected cultures

• Kinase Activity Assays:

  • Measures functional output of ERK1/2 activation

  • Can employ recombinant substrates or specific peptides

  • Provides quantitative measurement of kinase activity

• Reporter Gene Assays:

  • Uses ERK1/2-responsive promoter elements linked to reporter genes

  • Provides integrated measure of pathway activation over time

  • Useful for high-throughput screening of inhibitors or mutants

When studying O1 protein function specifically, it's crucial to compare wild-type virus with O1L deletion mutants and to examine both transient and sustained ERK1/2 activation patterns, as O1 protein appears to be particularly important for the sustained phase of activation .

How should researchers interpret differences in ERK1/2 activation patterns between wild-type and O1L-deleted viruses?

When analyzing ERK1/2 activation patterns, researchers should consider:

• Temporal Dynamics: Wild-type viruses with intact O1L gene (like CVA) induce sustained ERK1/2 activation, while O1L deletion mutants (CVA-ΔO1L) show only transient activation . The key difference is not in the initial activation but in the maintenance of activation over time.

• Interpretation Framework:

  • Early activation (0-2 hours post-infection): Primarily mediated by VGF and likely independent of O1 protein

  • Sustained activation (2-24 hours post-infection): Requires O1 protein function

  • Amplitude vs. duration: Assess both the strength (fold-change from baseline) and persistence of signaling

• Statistical Analysis:

  • Use repeated measures ANOVA for time-course data

  • Calculate area under the curve (AUC) for activation profiles to quantify sustained activation

  • Employ appropriate normalization to account for differences in infection efficiency

• Biological Context:

  • Connect ERK1/2 activation patterns with downstream biological outcomes such as:

    • Virus replication efficiency

    • Cytopathic effect (CPE)

    • Plaque size

    • Virulence in animal models

• Controls and Validations:

  • Include pharmacological controls (MEK inhibitors) to confirm specificity

  • Verify that differences result from O1L deletion rather than other factors by testing revertant viruses (where O1L is reintroduced)

This interpretation approach helps distinguish O1 protein's specific contribution to ERK1/2 signaling from the contributions of other viral factors like VGF, providing insights into the mechanisms of poxvirus modulation of host signaling pathways.

What bioinformatic approaches can predict O1 protein interactions with host cell factors?

Several bioinformatic approaches can help predict O1 protein interactions with host cell factors:

• Sequence-Based Prediction Methods:

  • Motif Scanning: Identify known interaction motifs (e.g., SH2/SH3 binding sites, kinase recognition motifs) within the O1 protein sequence

  • Domain Prediction: Use tools like SMART, Pfam, or InterPro to identify functional domains that might mediate protein-protein interactions

  • Disorder Prediction: Identify intrinsically disordered regions that often serve as protein-protein interaction sites

• Structure-Based Approaches:

  • Homology Modeling: Generate structural models based on related proteins with known structures

  • Molecular Docking: Perform in silico docking of O1 protein models with potential interaction partners

  • CoVES Methodology: Apply structure-based residue preferences to predict functional interactions

• Network-Based Predictions:

  • Interolog Mapping: Transfer known protein-protein interactions from homologous proteins in other systems

  • Functional Association Networks: Use databases like STRING to identify proteins functionally associated with known O1 targets

  • Pathway Analysis: Identify potential interaction partners based on known components of the Raf/MEK/ERK pathway

• Machine Learning Approaches:

  • Interface Prediction: Train machine learning models to recognize protein-protein interaction interfaces

  • Integration of Multiple Features: Combine sequence, structure, and evolutionary information to improve prediction accuracy

• Evolutionary Analysis:

  • Co-evolutionary Analysis: Identify correlated mutations between O1 protein and potential partners

  • Conservation Mapping: Identify highly conserved surface patches likely to mediate functional interactions

These computational predictions should be validated experimentally using techniques such as co-immunoprecipitation, proximity labeling (BioID), or yeast two-hybrid screening to confirm predicted interactions.

What are the most promising research avenues for therapeutic targeting of O1 protein function?

Several promising research avenues for therapeutic targeting of O1 protein function include:

• Small Molecule Inhibitors:

  • Develop inhibitors that specifically disrupt O1 protein's ability to sustain ERK1/2 activation

  • These could potentially attenuate poxvirus virulence without affecting viral antigen expression

  • High-throughput screening of compound libraries against recombinant O1 protein function

  • Structure-based drug design once detailed structural information becomes available

• Peptide-Based Inhibitors:

  • Design peptides that mimic interaction interfaces between O1 protein and its binding partners

  • Cell-penetrating peptides could be developed to deliver the inhibitory sequences intracellularly

  • These might offer higher specificity than small molecule approaches

• Therapeutic Antibodies:

  • Generate antibodies that specifically recognize and neutralize O1 protein

  • These could be useful for post-exposure prophylaxis for poxvirus infections

  • Humanized or fully human antibodies would be required for clinical applications

• Gene-Targeted Approaches:

  • Antisense oligonucleotides or RNAi targeting O1L mRNA

  • CRISPR-based approaches to disrupt O1L gene expression in infected cells

• Host-Directed Therapies:

  • Target host factors that interact with O1 protein rather than the viral protein itself

  • This approach might have a higher barrier to resistance development

  • Could potentially be effective against multiple poxviruses due to the conserved nature of O1 protein

The finding that O1 protein deletion reduces virulence and spread in mice while still allowing viral gene expression suggests that O1 protein inhibitors might serve as effective antivirals that attenuate disease without preventing the development of protective immunity, potentially useful for therapeutic vaccines.

How might our understanding of O1 protein function inform vaccine development strategies?

Understanding O1 protein function can significantly impact vaccine development strategies in several ways:

• Attenuated Vaccine Design:

  • Targeted modification of O1L gene could create rationally attenuated vaccine strains

  • The reduced virulence and spread observed in O1L deletion mutants while maintaining immunogenicity makes this approach promising

  • Fine-tuning of O1L function through partial deletions or point mutations could optimize the balance between safety and immunogenicity

• Vector Improvement:

  • MVA already lacks functional O1L gene (fragmented ORF) and is widely used as a vaccine vector

  • Understanding the role of O1 protein in different cellular contexts could help optimize vector tropism and expression characteristics

  • Engineering chimeric O1 proteins with selective functionality could enhance vector performance in specific applications

• Adjuvant Development:

  • Since O1 protein modulates the Raf/MEK/ERK pathway , recombinant O1 protein variants could potentially be developed as immunomodulatory adjuvants

  • Controlled activation of ERK1/2 signaling in antigen-presenting cells could enhance vaccine responses

• Safety Enhancement:

  • Knowledge of O1 protein's role in virulence allows for more precise safety assessment of vaccine candidates

  • Monitoring O1L gene integrity could be an important quality control parameter for attenuated vaccines

• Multivalent Vaccine Platforms:

  • Understanding how O1 protein affects antigen presentation and immune responses could inform the design of multivalent vaccines

  • Differential O1 protein function in various cell types might be exploited to direct immune responses toward specific pathways

The experience with MVA, which contains a fragmented O1L ORF and shows excellent safety while maintaining immunogenicity , demonstrates the practical relevance of O1 protein studies for vaccine development. Further research into the precise mechanisms by which O1 protein modulates host responses could enable even more sophisticated vaccine design strategies.