Recombinant Vaccinia virus Uncharacterized protein VACWR005 (VACWR005)

What is VACWR005 and where is it encoded in the Vaccinia virus genome?

VACWR005 is an uncharacterized protein encoded by the Vaccinia virus Western Reserve strain. Based on genomic annotations, it consists of 48 amino acids with the sequence: MYDDLIEQCH LSMERKSKLV DKALNKLEST IGQSRLSYLP PEIMRNII . This small protein is encoded early in the vaccinia virus genome, though its precise genomic location and relationship to other viral genes requires further characterization. Unlike well-characterized vaccinia proteins such as K4L (which functions as a nicking-joining enzyme) or protein 169 (a translation inhibitor), the function of VACWR005 remains largely unknown .

What expression systems are available for producing recombinant VACWR005 protein?

Recombinant VACWR005 can be produced in multiple expression systems, each with distinct advantages for different research applications:

For specialized applications, the protein can also be produced with tags such as the Avi-tag for in vivo biotinylation in E. coli, which enables selective labeling for detection and purification purposes .

How can VACWR005 be purified for experimental studies?

The purification of VACWR005 typically follows standard protein purification protocols with modifications specific to the expression system and tags used. For lyophilized recombinant protein, reconstitution in deionized sterile water is recommended to achieve the desired concentration . The purification strategy should include:

• Initial capture using affinity chromatography based on the specific tag (His-tag, GST, etc.)

• Intermediate purification using ion exchange chromatography

• Polishing steps such as size exclusion chromatography to achieve >85% purity as verified by SDS-PAGE

For recombinant VACWR005 expressed with biotinylation tags, streptavidin-based affinity purification provides a highly specific method to isolate the protein with minimal contaminants .

What approaches can be used to determine the subcellular localization of VACWR005 during infection?

Determining the subcellular localization of VACWR005 requires multiple complementary approaches:

• Immunofluorescence microscopy: Using antibodies against VACWR005 or against fusion tags in recombinant constructs, researchers can visualize its distribution. Based on patterns observed with other vaccinia proteins, it may be important to determine if VACWR005 is excluded from virus factories like protein 169 .

• Cellular fractionation: Biochemical separation of nuclear, cytoplasmic, membrane, and organelle fractions followed by western blotting can confirm microscopy findings.

• Time-course analysis: Examination of localization changes throughout the viral infection cycle, particularly during early and late phases.

• Co-localization studies: Double immunofluorescence with markers for different cellular compartments and viral structures.

The pattern of localization can provide significant insights into function, as demonstrated with protein 169, which was found to be cytoplasmic and excluded from virus factories, correlating with its role in translation inhibition .

What computational approaches and tools are most effective for predicting potential functions of VACWR005?

For an uncharacterized protein like VACWR005, computational prediction can guide experimental design:

• Sequence homology analysis: Compare VACWR005 against protein databases using BLAST, HHpred, and HMMER to identify distant homologs.

• Structural prediction: Use AlphaFold2, I-TASSER, or Rosetta to generate structural models, which may reveal functional domains.

• Protein interaction prediction: Tools such as STRING and InterProScan can identify potential binding partners and functional associations.

• Motif scanning: Scan for functional motifs using PROSITE, PFAM, and SMART databases.

• Expression correlation analysis: Analyze transcriptomic data to identify genes with similar expression patterns during infection.

These computational approaches should be integrated to develop testable hypotheses about VACWR005 function, similar to how functions of other vaccinia proteins were initially hypothesized before experimental verification .

How might VACWR005 contribute to vaccinia virus immune evasion strategies?

While VACWR005's function is unknown, vaccinia virus encodes numerous proteins that modulate host immune responses. To investigate potential immune evasion functions:

• Host protein interaction studies: Identify host proteins that interact with VACWR005 using yeast two-hybrid, co-immunoprecipitation followed by mass spectrometry, or proximity labeling techniques.

• Effect on immune signaling pathways: Examine the impact of VACWR005 expression on NFκB, IRF3, STAT, and other immune signaling pathways using reporter assays.

• Viral deletion mutant phenotyping: Compare the replication of wild-type virus versus VACWR005 deletion mutants in immune-competent versus immune-deficient systems.

• Cytokine profiling: Measure changes in pro-inflammatory cytokine and chemokine production during infection with viruses expressing or lacking VACWR005.

Similar approaches revealed that protein 169 inhibits host protein synthesis and thereby broadly suppresses innate immune responses, despite being dispensable for virus replication in cell culture .

What is the optimal approach for generating a VACWR005 deletion mutant to study its function?

Creating a VACWR005 deletion mutant requires careful consideration of several factors:

• Deletion strategy: Complete gene deletion versus functional domain disruption, considering potential overlapping reading frames or regulatory elements.

• Recombination methodology: Homologous recombination using a transfer plasmid with flanking sequences and a selection marker, similar to methods used for creating other vaccinia virus mutants .

• Confirmation methods:

  • PCR verification of deletion

  • Sequencing of the modified region

  • Western blotting to confirm absence of protein expression

  • Next-generation sequencing to confirm no unintended mutations

• Control viruses: Generation of revertant viruses where the gene is reintroduced to ensure phenotypic differences are specifically due to VACWR005 deletion.

As observed with the K4L deletion virus, careful phenotypic comparison between wild-type, deletion, and revertant viruses is essential to accurately attribute functional changes to the deleted gene .

How should researchers design protein-protein interaction studies to identify VACWR005 binding partners?

To identify VACWR005 interaction partners:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express tagged VACWR005 in either infected cells or appropriate expression systems

  • Perform pulldowns under varying conditions (detergent, salt concentration)

  • Analyze by LC-MS/MS to identify co-purifying proteins

  • Use quantitative proteomics with SILAC or TMT labeling for increased specificity

• Proximity-dependent labeling:

  • Create BioID or TurboID fusions with VACWR005

  • Express in cells during infection

  • Identify proximal proteins through streptavidin pulldown and MS

• Yeast two-hybrid screening:

  • Use VACWR005 as bait against human cDNA libraries or vaccinia virus ORFeome

  • Validate interactions through secondary assays

• Criteria for identifying significant interactions:

  • Statistical enrichment over controls

  • Reproducibility across biological replicates

  • Validation by orthogonal methods

Such approaches have successfully identified functional partners for other vaccinia virus proteins, revealing their roles in cellular processes .

What are the key considerations for studying the temporal expression pattern of VACWR005 during infection?

Understanding when VACWR005 is expressed provides important clues to its function:

• Time-course analysis:

  • Infect cells with vaccinia virus at high MOI

  • Collect samples at various timepoints (0, 0.5, 1, 2, 4, 8, 12, 24 hours post-infection)

  • Analyze by RT-qPCR for mRNA and western blotting for protein levels

• Promoter characterization:

  • Identify the putative promoter region of VACWR005

  • Create reporter constructs to assess temporal activity

  • Compare with known early, intermediate, and late promoters

• Dependence on viral DNA replication:

  • Treat infected cells with cytosine arabinoside to block viral DNA replication

  • Determine if VACWR005 expression is affected, classifying it as an early or post-replicative gene

• Co-expression analysis:

  • Compare VACWR005 expression timing with functionally related genes

  • Perform clustering analysis of temporal expression data

Similar temporal expression studies with protein 169 revealed its early expression during infection, providing insights into its role in host translation inhibition .

How should researchers analyze potential structural features of VACWR005 to generate functional hypotheses?

A comprehensive structural analysis of VACWR005 should include:

• Secondary structure prediction:

  • Use multiple prediction algorithms (PSIPRED, JPred, SOPMA)

  • Identify potential α-helices, β-sheets, and disordered regions

  • Compare predictions with known vaccinia virus protein structures

• Tertiary structure modeling:

  • Generate models using AlphaFold2 and other protein structure prediction tools

  • Validate models through energy minimization and Ramachandran plot analysis

  • Compare structural features with known protein folds

• Functional site prediction:

  • Identify potential binding pockets, catalytic sites, or interaction interfaces

  • Predict post-translational modification sites

  • Map conserved residues onto the structural model

• Experimental validation:

  • Design site-directed mutagenesis experiments targeting predicted functional residues

  • Express recombinant variants for functional testing

  • Perform protein-fragment complementation assays for interaction domains

Analysis of other vaccinia virus proteins like the K4L nicking-joining enzyme demonstrates how structural features can reveal enzymatic functions .

What approaches can differentiate between direct and indirect effects when studying VACWR005's impact on viral replication?

Distinguishing direct from indirect effects requires multiple experimental approaches:

• Complementation studies:

  • Create VACWR005 deletion virus

  • Complement with wild-type or mutant versions of the protein

  • Assess which specific functions are restored

• Time-of-addition experiments:

  • Add recombinant VACWR005 at different stages of infection

  • Determine when the protein must be present to observe its effects

• Dose-dependency analysis:

  • Express varying levels of VACWR005 using inducible systems

  • Establish quantitative relationships between protein levels and phenotypic effects

• Mechanistic dissection:

  • Identify potential mechanistic pathways through which VACWR005 might function

  • Systematically inhibit each pathway and observe effects on VACWR005 function

  • Use genetic screening approaches to identify suppressors or enhancers

This multi-faceted approach is similar to how researchers determined that protein 169's effects on host immunity were directly mediated through translation inhibition rather than through other mechanisms .

How can researchers effectively compare VACWR005 to other uncharacterized poxvirus proteins to establish functional relationships?

Comparative analysis of VACWR005 with other poxvirus proteins should include:

• Comparative genomics:

  • Identify orthologs in related poxviruses

  • Analyze sequence conservation and divergence

  • Create phylogenetic trees to infer evolutionary relationships

• Conserved domain analysis:

  • Map domains shared between VACWR005 and characterized proteins

  • Identify conservation patterns in specific functional regions

  • Calculate selection pressures on different protein regions

• Co-evolution network analysis:

  • Identify proteins that co-evolve with VACWR005 across poxvirus species

  • Construct functional networks based on co-evolution patterns

  • Infer functional relationships from network topology

• Comparative transcriptomics and proteomics:

  • Compare expression patterns across related viruses

  • Identify consistently co-expressed genes across species

  • Analyze protein abundance correlation patterns

This comparative approach could reveal whether VACWR005 functions similarly to other vaccinia proteins like K4L or protein 169, which have distinct roles in viral replication and host interaction .

What are the most promising research directions for elucidating VACWR005 function in vaccinia virus biology?

Based on current knowledge of vaccinia virus proteins, several promising research directions for VACWR005 include:

• Role in viral replication: Determine if VACWR005 affects viral DNA replication, transcription, or virion assembly through deletion mutant analysis and replication assays.

• Host-pathogen interactions: Investigate whether VACWR005 interacts with host factors to modulate cellular functions like protein 169 does with translation machinery .

• Immunomodulatory functions: Assess whether VACWR005 affects innate or adaptive immune responses, particularly since many vaccinia proteins function as immune evasion factors.

• Structural biology approach: Determine the three-dimensional structure of VACWR005 to gain insights into its potential function.

• Systems biology integration: Position VACWR005 within the broader context of vaccinia virus infection through network analysis and multi-omics approaches.

Combining these approaches with careful experimental design and validation will likely provide significant insights into this uncharacterized protein's role in vaccinia virus biology.

How might characterization of VACWR005 contribute to the development of improved recombinant vaccinia virus vectors?

Understanding VACWR005 could enhance recombinant vaccinia virus vector development through:

• Vector optimization: If VACWR005 affects vector properties like immunogenicity or stability, its modification could improve vaccine performance.

• Safety profile enhancement: Determining whether VACWR005 contributes to virulence could help create safer attenuated vectors, similar to the development of MVA and NYVAC strains .

• Expression level modulation: If VACWR005 influences gene expression, manipulating it could enhance foreign antigen expression in recombinant vaccines.

• Vector adaptability: Understanding VACWR005's role might reveal ways to adapt vaccinia vectors for specific tissues or applications.

• Polyvalent vaccine design: Knowledge of VACWR005 could inform the development of polyvalent vaccines that express multiple foreign antigens, leveraging vaccinia's large capacity for foreign DNA .