Recombinant African swine fever virus Uncharacterized protein DP60R (BA71V-158)

What is the DP60R protein and what are its basic characteristics?

The DP60R protein (BA71V-158) is an uncharacterized protein from African swine fever virus with a full length of 60 amino acids. The complete amino acid sequence is MSSIWPPQKKVFTVGFITGGVTPVMVSFVWPAAQPQKKINYSRKKKYFRPRSFYKKNVSF . This protein has been identified as a new variable region in ASFV, with clustering analyses showing similar patterns to other genetic determinants within the virus . The protein is typically expressed in E. coli systems for research purposes with an N-terminal His-tag to facilitate purification and downstream applications .

The DP60R protein belongs to a group of ASFV proteins that remain functionally uncharacterized but may play significant roles in viral replication, host interaction, or virulence. Understanding these basic characteristics provides a foundation for more targeted investigations into the protein's biological significance and potential as a research target.

What are the optimal storage and handling conditions for recombinant DP60R protein?

When working with recombinant DP60R protein, researchers should follow specific storage and handling protocols to maintain protein integrity. The recombinant protein is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C upon receipt . For optimal results, brief centrifugation of the vial before opening is recommended to bring the contents to the bottom .

For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To prevent degradation during long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being standard practice) and aliquot before storing at -20°C/-80°C . This prevents protein degradation from repeated freeze-thaw cycles, which should be avoided whenever possible. For working aliquots, storage at 4°C for up to one week is acceptable . These handling procedures are essential for maintaining protein stability and ensuring experimental reproducibility.

How can researchers verify the purity and integrity of recombinant DP60R protein?

Verification of recombinant DP60R protein purity and integrity involves multiple analytical techniques. SDS-PAGE analysis should be the first method employed, as commercial preparations typically guarantee greater than 90% purity by this method . When performing SDS-PAGE, use appropriate molecular weight markers, as the DP60R protein with His-tag has a predicted molecular weight of approximately 7-8 kDa.

Additional verification methods should include:

For functional integrity verification, binding assays or activity tests specific to your research context may be necessary. Although DP60R is currently uncharacterized, interaction studies with other viral or cellular proteins can provide evidence of proper folding and functional capacity. These verification steps are critical before proceeding with downstream experimental applications.

What expression systems are most effective for producing recombinant DP60R protein?

The table below compares different expression systems for DP60R protein production:

For most applications, E. coli remains the system of choice due to its balance of yield and cost-effectiveness. When using E. coli expression, optimizing induction conditions (IPTG concentration, temperature, duration) and lysis procedures significantly impacts the quality of the final product. While commercial sources typically use E. coli systems with N-terminal His-tags , researchers may need to explore alternative tags or fusion partners if protein solubility or activity is compromised.

How can researchers design experiments to investigate potential roles of DP60R in ASFV virulence?

Investigating DP60R's potential role in ASFV virulence requires a multi-faceted experimental approach. The DP60R gene has been identified as a variable region in ASFV with possible involvement in virulence mechanisms, similar to other characterized ASFV genes like UK (DP96R) .

A comprehensive experimental design strategy should include:

• Gene Knockout/Mutation Studies: Generate recombinant ASFV strains with DP60R deletions or mutations using genetic manipulation techniques similar to those used for other ASFV virulence determinants . Compare the pathogenicity of these modified strains with wild-type virus in suitable in vitro and in vivo models.

• Protein Interaction Mapping: Identify potential binding partners of DP60R using techniques such as co-immunoprecipitation, yeast two-hybrid screening, or proximity labeling methods. This will help establish the protein's interaction network within viral and host cellular contexts.

• Functional Assays: Develop assays to measure specific virulence-related parameters (e.g., host cell invasion, replication efficiency, immune evasion) in the presence and absence of functional DP60R protein.

• Structural Analysis: Determine the three-dimensional structure of DP60R using X-ray crystallography or NMR to gain insights into potential functional domains that might contribute to virulence.

• Host Response Analysis: Evaluate host immune responses to wild-type versus DP60R-modified viruses, including transcriptomic and proteomic profiling of infected cells.

This multi-dimensional approach allows researchers to establish whether DP60R functions as a virulence determinant and, if so, the mechanisms through which it contributes to ASFV pathogenicity. The findings could potentially inform rational design of attenuated vaccine candidates, similar to other ASFV virulence genes that have been manipulated for vaccine development .

What sequencing methodologies are most appropriate for analyzing DP60R genetic variants?

Multiple sequencing approaches can be employed to analyze DP60R genetic variants, each with specific advantages depending on research objectives. Based on ASFV research methodologies, these approaches include:

When specifically analyzing DP60R genetic variants, researchers have successfully employed conventional PCR with region-specific primer pairs followed by Sanger sequencing for typing field samples . For more comprehensive analysis, Illumina NovaSeq 6000 sequencing with PE150 mode has been used for whole-genome ASFV sequencing, which would include the DP60R region .

For experimental design, consider that ASFV genomes are relatively large and may have unfavorable virus/host-ratio in field samples, necessitating scaled-up sequencing efforts . DNA extraction quality is critical, with a minimum of 100 ng of DNA typically required for commercial sequencing services . Analysis should incorporate appropriate bioinformatics pipelines to identify variants, with special attention to quality filtering and alignment parameters suited to the ASFV genome characteristics.

How can contradictions in DP60R characterization data be systematically analyzed and resolved?

When studying uncharacterized proteins like DP60R, researchers may encounter contradictory data across different studies or experimental approaches. Systematic analysis and resolution of these contradictions is essential for accurate characterization. Drawing from approaches used in bioinformatics and policy analysis fields, researchers can implement the following framework:

• Ontology-Based Classification: Develop a standardized ontology for DP60R-related data to ensure that contradictions are not simply the result of different semantic levels of description . For example, contradictions might arise when researchers describe protein functions at different levels of granularity (molecular interaction vs. cellular effect).

• Contradiction Mapping: Identify potential contradictions using automated tools similar to PolicyLint, which detects logical contradictions in data sets . For DP60R, this would involve comparing findings across multiple studies and experimental approaches, specifically looking for:

  • Functional contradiction (e.g., pro-virulence vs. anti-virulence effects)

  • Structural contradiction (e.g., different predicted protein structures)

  • Localization contradiction (e.g., different subcellular locations)

• Experimental Validation: Design targeted experiments to directly address identified contradictions:

• Context-Dependency Analysis: Determine whether contradictions reflect genuine biological variability rather than experimental error. For DP60R, this might involve testing the protein's function across different ASFV strains, host cell types, or infection stages.

• Bayesian Integration Framework: Implement a statistical approach that weighs contradictory evidence based on methodological strength, sample size, and reproducibility to derive the most probable biological reality.

This systematic approach not only resolves contradictions but potentially reveals important biological insights about context-dependent functions of DP60R that might be relevant to ASFV pathogenesis and vaccine development strategies.

What are the implications of DP60R structure-function relationships for rational vaccine design?

Understanding the structure-function relationships of DP60R has significant implications for rational vaccine design against ASFV. Although DP60R remains largely uncharacterized, insights can be drawn from studies of other ASFV proteins that have been successfully used in vaccine development strategies.

ASFV gene manipulation has become a standard method for developing experimental live attenuated vaccine strains . Similar to the UK (DP96R) gene, which was identified as a determinant of virulence in ASFV, understanding the structural and functional aspects of DP60R could potentially reveal its role in viral pathogenesis . This knowledge would inform whether DP60R is a suitable target for genetic manipulation in vaccine development.

The structure-function analysis of DP60R should consider:

• Epitope Mapping: Identification of immunogenic regions within DP60R that could elicit protective immune responses. This requires:

  • Computational prediction of B-cell and T-cell epitopes

  • Experimental validation through epitope mapping techniques

  • Assessment of epitope conservation across ASFV strains

• Functional Domain Analysis: Determination of functional domains that might contribute to virulence and could be targets for attenuation:

  • Protein interaction domains

  • Enzymatic activity sites

  • Host cell targeting signals

• Variability Assessment: Analysis of genetic variability of DP60R across ASFV isolates to determine:

  • Conserved regions suitable as broad-spectrum vaccine targets

  • Variable regions that might affect vaccine efficacy against different strains

  • Potential for recombination events that could impact vaccine stability

• Immunomodulatory Potential: Investigation of whether DP60R influences host immune responses, which could affect its suitability as a vaccine component.

The identification of DP60R as a new variable region suggests it may contribute to strain-specific characteristics of ASFV. If structural and functional studies reveal a role in virulence, similar to other characterized ASFV proteins, DP60R could become a target for rational attenuation to develop live attenuated vaccine candidates. Alternatively, if specific epitopes are identified, these could be incorporated into subunit or vectored vaccine approaches.

How can high-throughput methodologies be optimized for comprehensive DP60R interaction network mapping?

Mapping the comprehensive interaction network of DP60R requires optimized high-throughput methodologies that can identify viral-viral and viral-host protein interactions with high confidence. Based on advanced molecular biology techniques and specific considerations for ASFV proteins, researchers should consider the following optimized approaches:

• Proximity-Based Labeling Technologies:

  • BioID or TurboID fusion constructs with DP60R to label proximal proteins in relevant cellular contexts

  • APEX2-based proximity labeling for temporal resolution of interactions during infection

  • Optimization parameters should include expression levels, labeling time, and subcellular targeting

• Affinity Purification-Mass Spectrometry (AP-MS) Workflows:

• Yeast-Based Interaction Screens:

  • Modified split-ubiquitin membrane yeast two-hybrid for membrane-associated interactions

  • Yeast three-hybrid systems to detect RNA-mediated protein interactions

  • Adaptation of systems for potential toxicity of viral proteins

• Protein Complementation Assays in Relevant Cell Types:

  • NanoBiT, split-GFP, or DHFR-based complementation assays

  • Live-cell imaging to capture dynamic interaction networks

  • Implementation in porcine macrophage cell lines for physiological relevance

• Computational Integration Framework:

  • Machine learning algorithms to predict interactions based on structural features

  • Network analysis to prioritize high-confidence interactions

  • Integration with existing ASFV protein interaction datasets

When optimizing these methods specifically for DP60R, researchers should consider its small size (60 amino acids) , potential for forming multimeric structures, and possible post-translational modifications. The experimental designs should include both systems with recombinant expression of DP60R and, when possible, analyses in the context of ASFV infection to capture physiologically relevant interactions.

Additionally, validation of key interactions through orthogonal methods (co-immunoprecipitation, FRET/BRET, or co-localization studies) is essential to establish a high-confidence interaction network that can inform functional studies and potential therapeutic targeting strategies.

How can DP60R genetic variation data be integrated into ASFV evolution and epidemiology models?

The integration of DP60R genetic variation data into ASFV evolution and epidemiology models requires a sophisticated approach that connects molecular-level genetic changes to broader patterns of viral spread and virulence. Recent research has identified DP60R as a new variable region in ASFV, with clustering analyses providing valuable insights into viral evolution . This emerging knowledge can be incorporated into comprehensive models through several methodological approaches:

• Phylogenomic Integration: Researchers should sequence the DP60R gene across diverse ASFV isolates using both targeted Sanger sequencing for specific analysis and whole-genome approaches using platforms like NovaSeq 6000 for comprehensive coverage . The resulting sequence data should be incorporated into phylogenetic analyses to:

  • Identify DP60R variant clusters that correlate with geographical distribution

  • Determine whether DP60R variations align with established ASFV genotypes

  • Calculate evolutionary rates specific to the DP60R region compared to the whole genome

• Structure-Function Correlation: Genetic variations in DP60R should be mapped to protein structure predictions to assess potential functional impacts:

  • Non-synonymous mutations should be evaluated for effects on protein folding and stability

  • Variations in potential functional domains should be prioritized for experimental validation

  • Selection pressure analyses (dN/dS ratios) should be calculated to identify regions under positive selection

• Epidemiological Correlation Analysis: Statistical frameworks should be developed to correlate specific DP60R variants with epidemiological parameters:

• Integrated Modeling Framework: Develop computational models that incorporate DP60R variation data alongside other genetic and environmental factors:

  • Bayesian phylogeographic models to reconstruct viral spread patterns

  • Machine learning approaches to predict virulence based on genetic signatures

  • Network models of transmission incorporating both viral genetic factors and host/environmental variables

This integrated approach will allow researchers to determine whether DP60R variations contribute significantly to ASFV evolution, potentially identifying specific mutations that correlate with changes in virulence, host range, or geographical distribution. Such knowledge would inform surveillance strategies, vaccine development, and outbreak response measures, particularly as ASFV continues to diverge into distinct lineages with different variants characterized by high-impact mutations .

What methodological approaches can reveal the functional significance of post-translational modifications in DP60R?

Investigating post-translational modifications (PTMs) of DP60R requires specialized methodological approaches that can identify, characterize, and determine the functional significance of these modifications. While the basic recombinant protein expressed in E. coli lacks eukaryotic PTMs , the native DP60R protein in the context of ASFV infection may undergo various modifications that affect its function, localization, or interactions.

A comprehensive methodological framework includes:

• PTM Identification and Mapping:

  • Mass Spectrometry Approaches:

    • High-resolution LC-MS/MS with enrichment strategies specific for phosphorylation, glycosylation, ubiquitination, and other PTMs

    • Top-down proteomics to analyze intact DP60R protein and identify combinatorial PTM patterns

    • SWATH-MS for quantitative analysis of modification stoichiometry

  • Site-specific antibodies against predicted modification sites for immunological detection

• Temporal Dynamics Analysis:

  • Pulse-chase labeling combined with immunoprecipitation to track modification kinetics

  • Time-resolved proteomics during ASFV infection cycle

  • Development of biosensors for real-time visualization of specific PTM events

• Functional Significance Assessment:

• Structural Impact Assessment:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine conformational changes induced by PTMs

  • NMR spectroscopy to analyze structural dynamics of modified protein

  • Molecular dynamics simulations to predict PTM effects on protein structure and flexibility

• Biological Context Validation:

  • CRISPR-Cas9 modification of PTM sites in the viral genome

  • Generation of recombinant viruses with PTM-deficient DP60R

  • Infection studies in relevant cell types comparing wild-type and PTM-mutant viruses

When implementing these methodologies for DP60R specifically, researchers should consider its small size (60 amino acids) and the amino acid composition (MSSIWPPQKKVFTVGFITGGVTPVMVSFVWPAAQPQKKINYSRKKKYFRPRSFYKKNVSF) , which contains multiple potential modification sites, including serines and threonines for phosphorylation and lysines for ubiquitination or acetylation. The high proportion of basic residues (K and R) also suggests potential regulation through methylation.

Understanding the PTM landscape of DP60R will provide insights into its regulation during infection and may identify novel targets for therapeutic intervention against ASFV.