Recombinant African swine fever virus Protein MGF 110-13L (Mal-018)

What is MGF 110-13L protein and what is its role in African swine fever virus?

MGF 110-13L is a protein encoded by the African swine fever virus (ASFV) and belongs to the multigene family (MGF) 110. Research has identified MGF_110-13L as an immunogenic protein that can induce antibody responses in infected animals . The MGF 110 family consists of thirteen highly diverse paralogs among ASFV genomes, with the ASFV-G genome containing 11 different MGF110 family genes . Within this family, MGF 110-13L is expressed as a glycosylated homodimer in eukaryotic cells, exhibiting strong antigenic properties .

While the precise function of MGF 110-13L remains under investigation, research suggests it may play a role in viral pathogenesis. Studies have shown that MGF 110-13L-specific monoclonal antibodies can recognize the protein in lysates of ASFV-infected cells, indicating its expression during active infection . Interestingly, the MGF 110 genes have been considered targets for deletion in the development of live-attenuated vaccine candidates, suggesting their potential role in virulence .

How is MGF 110-13L protein typically expressed and purified for research purposes?

For research applications, MGF 110-13L protein can be expressed in both prokaryotic and eukaryotic expression systems, with the choice depending on the specific research objectives:

Prokaryotic Expression:

• Common system: E. coli-based expression of the full-length protein (1-160 amino acids) or specific domains

• Tags: Frequently expressed with His-tags to facilitate purification

• Purification: Typically involves affinity chromatography using nickel or cobalt resins

• Storage: Often lyophilized and stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Reconstitution: Generally reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage at -20°C/-80°C

Eukaryotic Expression:

• For studying native properties such as glycosylation, mammalian cell expression systems are preferred

• The main outer-membrane domain can be expressed separately for immunological studies

• Purified protein may exhibit two main bands by SDS-PAGE analysis with the higher molecular weight band being sensitive to PNGase F treatment, confirming glycosylation

When working with the recombinant protein, it's important to avoid repeated freeze-thaw cycles to maintain protein integrity and function .

What are the known structural features and post-translational modifications of MGF 110-13L?

MGF 110-13L exhibits several important structural features that contribute to its function:

Protein Structure:

• The full-length protein consists of 160 amino acids

• Contains predicted transmembrane domains, with functional studies focusing on the outer-membrane domain

• Forms a glycosylated homodimer in its native state when expressed in eukaryotic cells

Post-translational Modifications:

• N-glycosylation: The protein contains a predicted N-glycosylation site

• When purified from eukaryotic expression systems, MGF 110-13L shows two main bands by SDS-PAGE

• The higher molecular weight band is sensitive to PNGase F treatment, confirming glycosylation

Epitope Regions:

• Two linear epitopes have been identified using monoclonal antibodies:

  • Epitope 8C3: 48WDCQDGICKNKITESRFIDS67

  • Epitope 10E4: 122GDHQQLSIKQ131

• Both epitopes are highly conserved (59/59) among full-length MGF 110-13L protein sequences

• Epitope 10E4 has been identified as antigenic and potentially useful for serological diagnostic test development

Understanding these structural features is essential for designing effective detection methods and evaluating the protein's potential as a diagnostic or vaccine target.

How can CRISPR/Cas9 be utilized to study the function of MGF 110-13L in the context of ASFV infection?

CRISPR/Cas9 technology offers powerful approaches for investigating MGF 110-13L function:

Gene Deletion Protocol:

• Guide RNA Design: Design gRNAs targeting sequences flanking the MGF 110-13L gene

• Transfer Plasmid Construction: Create a plasmid containing homology arms for targeted recombination

• Transfection Procedure:

  • Infect macrophages with ASFV (e.g., 3 MOI)

  • Transfect with transfer plasmid (1.5 μg) and gRNA plasmids (0.75 μL each)

  • Use appropriate transfection reagent (e.g., Fugene HD)

  • Incubate at 37°C in 5% CO2 for 24h

• Mutant Isolation: Screen transfected cells for mutant virus using limiting dilution

• Verification: Confirm gene deletion by PCR and sequencing

Functional Assessment Methods:

• In Vitro Growth Kinetics: Compare replication efficiency between wild-type and mutant viruses

• Protein-Protein Interaction Studies: Identify potential binding partners using co-immunoprecipitation followed by mass spectrometry

• Transcriptomic Analysis: Evaluate host cell response changes using RNA-seq

• In Vivo Characterization: Assess pathogenicity, immunogenicity, and protective efficacy in animal models

This approach has been successfully applied to MGF 110-11L, leading to reduced pathogenicity while maintaining immunogenicity, suggesting similar approaches could reveal important functions of MGF 110-13L .

How does the sequence conservation of MGF 110-13L across different ASFV strains impact its utility as a diagnostic target?

The sequence conservation of MGF 110-13L has significant implications for its diagnostic potential:

Sequence Conservation Analysis:

• Among 191 MGF 110-13L protein sequences in the NCBI database (as of May 2023), 59 were full-length MGF 110-13L proteins

• Key epitope regions show remarkable conservation:

  • Epitope 8C3 (48WDCQDGICKNKITESRFIDS67): 100% conserved (59/59)

  • Epitope 10E4 (122GDHQQLSIKQ131): 100% conserved (59/59)

Diagnostic Applications Based on Conservation:

Potential Limitations:

• While MGF 110-13L protein shows high conservation, the gene can be present in different forms across strains:

  • Full-length (272-275 amino acids)

  • Mutants with deletion of the N-terminal outer membrane domain

  • Mutants with deletion of both N-terminal and transmembrane domains

• Diagnostic assays must account for potential structural variations while targeting the conserved epitopes

This high conservation makes the identified epitopes, particularly 10E4, excellent candidates for developing broadly applicable serological assays for ASFV diagnosis across different strains and geographical regions .

What role does MGF 110-13L play in ASFV pathogenesis and host immune evasion?

While the exact role of MGF 110-13L in ASFV pathogenesis remains under investigation, several lines of evidence suggest its potential functions:

Current Understanding:

• MGF 110-13L has been identified as an immunodominant antigen capable of inducing antibody responses in infected animals

• It is expressed during active ASFV infection, as demonstrated by the recognition of the protein in infected cell lysates by specific monoclonal antibodies

• The protein is glycosylated, suggesting potential roles in cell surface interactions or immune evasion

Functional Genomics Insights:

• The MGF 110 family, which includes MGF 110-13L, is part of the multigene families believed to be involved in host range and virulence

• Most ASFV proteins, including MGF 110-13L, have limited experimentally proven functions, representing a major gap in ASF research

• Understanding protein interactions (both viral-viral and viral-host) is crucial to elucidate function

Experimental Approaches to Determine Function:

• Gene Deletion Studies: Deletion of MGF 110-11L (another member of the MGF 110 family) resulted in reduced pathogenicity while maintaining immunogenicity, suggesting similar roles might exist for MGF 110-13L

• Protein-Protein Interaction Analyses: Techniques like yeast two-hybrid or co-immunoprecipitation followed by mass spectrometry could reveal binding partners

• Transcriptomic Analysis: Comparing host response to wild-type and MGF 110-13L-deleted viruses could provide insights into immune modulation functions

• In vitro Functional Assays: Purification of viral proteins for in vitro assays to confirm predicted functions

Research suggests that a comprehensive understanding of MGF 110-13L function could contribute to the development of rationally designed live-attenuated vaccines by identifying virulence factors that could be safely removed while maintaining immunogenicity .

How can transcriptomic approaches be used to understand the expression dynamics of MGF 110-13L during ASFV infection?

Transcriptomic approaches offer valuable insights into MGF 110-13L expression patterns and host responses:

RNA-Seq Methodology for ASFV Studies:

• Experimental Design:

  • Infect susceptible cells (e.g., porcine macrophages) with ASFV

  • Collect RNA samples at multiple time points post-infection (e.g., 0, 4, 8, 16, 24 hours)

  • Include samples from different ASFV strains (virulent vs. attenuated)

  • Prepare and sequence RNA libraries using standard protocols

• Specific Analyses for MGF 110-13L:

  • Temporal expression pattern throughout infection cycle

  • Correlation with other viral genes and host response genes

  • Comparison between virulent and attenuated strains

Previous Transcriptomic Findings from ASFV Research:

• RNAseq analysis identified 395 genes most differently expressed at euthanasia in highly pathogenic Georgia 2007 strain and 181 genes modified at 7 days post-infection with attenuated OURT88/3 strain

• Top differentially expressed host genes included macrophage markers, natural killer cell markers, chemokines, and immune response markers

• Such approaches could be applied specifically to understand MGF 110-13L expression dynamics

Integration with Proteomics:

• Combine transcriptomic data with proteomic approaches to confirm protein expression

• Mass spectrometry of purified ASFV particles has successfully identified viral and host-derived structural proteins

• This integrated approach could reveal post-transcriptional regulation mechanisms affecting MGF 110-13L

Understanding the expression dynamics of MGF 110-13L would provide valuable insights for vaccine development, potentially identifying when and how the protein contributes to virulence or immune evasion during infection.

What are the challenges and methodological approaches for studying protein-protein interactions involving MGF 110-13L?

Studying protein-protein interactions (PPIs) involving MGF 110-13L presents several challenges but is crucial for understanding its function in ASFV pathogenesis:

Key Challenges:

• Transmembrane Nature: MGF 110-13L contains transmembrane domains, making it difficult to study in solution

• Post-translational Modifications: Glycosylation may affect interaction partners

• Viral Context: Some interactions may only occur in the context of viral infection

• Technical Limitations: Standard PPI methods may not capture transient or weak interactions

• Limited Prior Knowledge: Lack of characterized interaction partners complicates validation

Methodological Approaches:

• Proximity Labeling Methods:

  • BioID: Fuse MGF 110-13L to a promiscuous biotin ligase (BirA*)

  • APEX2: Fuse to engineered ascorbate peroxidase

  • TurboID: Use for faster labeling kinetics

  • These methods allow for biotinylation of proteins in proximity to MGF 110-13L

• Yeast Two-Hybrid Screening:

  • Use the outer-membrane domain as bait

  • Screen against porcine macrophage cDNA libraries

  • Verify interactions using orthogonal methods

• Co-Immunoprecipitation with Specific Antibodies:

  • Utilize the generated monoclonal antibodies (8C3 and 10E4)

  • Perform pulldowns from infected cells

  • Identify interacting partners by Western blot or MS

• Protein Complementation Assays:

  • Split-GFP, split-luciferase, or NanoBiT systems

  • Test specific candidate interactions

  • Visualize interactions in living cells

Validation Approaches:

• Reciprocal pulldowns with tagged interacting partners

• Microscopy-based colocalization studies

• Functional assays to assess biological relevance of interactions

• Competition assays with synthetic peptides corresponding to identified epitopes

Understanding MGF 110-13L's interaction network would provide crucial insights into its role in ASFV pathogenesis. As noted in the research gap analysis, identifying the protein partners (both viral and host) for particular ASFV proteins represents a major gap in the field that could be addressed using these methodological approaches .

How can MGF 110-13L be utilized in developing next-generation ASFV vaccines?

MGF 110-13L offers several promising avenues for ASFV vaccine development:

Epitope-Based Vaccine Strategies:

• Utilize the highly conserved epitopes (48WDCQDGICKNKITESRFIDS67 and 122GDHQQLSIKQ131) in multi-epitope vaccine constructs

• Design chimeric proteins containing multiple protective ASFV epitopes

• Incorporate these epitopes into virus-like particles (VLPs) or nanoparticle platforms

Live-Attenuated Vaccine Development:

• Generate MGF 110-13L deletion mutants using CRISPR/Cas9 methodology

• Similar approaches with other MGF 110 family members have shown promise:

  • MGF 110-11L deletion resulted in reduced pathogenicity while maintaining immunogenicity

  • MGF 110-1L deletion has been studied in the context of ASFV-G (ASFV-G-ΔMGF110-1L)

Marker Vaccine Approaches:

• Develop DIVA (Differentiating Infected from Vaccinated Animals) vaccines by:

  • Deleting or modifying MGF 110-13L epitopes in attenuated strains

  • Using the epitope peptides in companion diagnostic assays

Experimental Design for Live-Attenuated Vaccine Evaluation:

Considerations for Optimal Vaccine Design:

• Attenuation Balance: Ensure sufficient attenuation for safety while maintaining immunogenicity

• Cross-Protection: Leverage the high conservation of MGF 110-13L epitopes across strains

• Immune Response Profiling: Characterize both humoral and cell-mediated responses

• Delivery Systems: Optimize administration routes and adjuvants

• Thermostability: Develop formulations suitable for field use in endemic regions

The development of effective ASFV vaccines remains a critical gap in controlling this devastating disease. MGF 110-13L offers promising avenues for both traditional live-attenuated approaches and next-generation epitope-based strategies .

How can MGF 110-13L research contribute to improved ASFV detection methods in field settings?

Research on MGF 110-13L has direct applications for developing enhanced ASFV detection methods for field use:

Epitope-Based Diagnostic Development:

• Pen-Side Lateral Flow Assays:

  • Utilize the highly conserved 10E4 epitope (122GDHQQLSIKQ131)

  • Design as conjugate with carrier proteins and gold nanoparticles

  • Optimize for sensitivity and specificity with field samples

  • Package as easy-to-use kits requiring minimal equipment

• Portable ELISA Systems:

  • Develop simplified ELISA protocols using epitope peptides

  • Adapt for battery-powered portable readers

  • Create lyophilized reagents for stability in field conditions

• Multiplex Field Tests:

  • Combine MGF 110-13L epitopes with other ASFV antigens

  • Design assays detecting multiple targets simultaneously

  • Incorporate internal controls for quality assurance

Performance Data from Epitope Testing:

Note: Based on dot blot assay using synthetic peptide EP8 (10E4 epitope)

Implementation Strategy for Field Settings:

• Field Validation:

  • Conduct multi-site trials in endemic regions

  • Compare against gold standard laboratory methods

  • Assess performance with diverse ASFV strains

  • Evaluate test stability under various environmental conditions

• Integration with Surveillance Systems:

  • Develop complementary mobile apps for result reporting

  • Create systems for geotagging samples

  • Link with regional diagnostic networks

  • Establish alert systems for outbreak detection

Advantages of MGF 110-13L-Based Field Tests:

• The high conservation of epitopes ensures broad detection capabilities across ASFV strains

• The strong reactivity with both experimental and field sera suggests good sensitivity

• Peptide-based tests offer higher stability compared to whole protein assays

• Simplified sample requirements make tests suitable for field veterinarians

These improved field detection methods would significantly enhance surveillance capabilities, particularly in resource-limited settings where laboratory infrastructure is limited, contributing to better control of ASF outbreaks.

How does MGF 110-13L compare structurally and functionally to other members of the MGF 110 family?

Understanding the similarities and differences between MGF 110-13L and other MGF 110 family members provides valuable insights into their specialized functions:

Structural Comparison:

Distribution and Gene Organization:

• The MGF 110 family consists of thirteen highly diverse paralogs among ASFV genomes

• ASFV-G genome contains 11 different MGF110 family genes (1L, 2L, 3L, 4L, 5L-6L, 7L, 9L, 10-14L, 12L, 13La, and 13Lb)

• Some genomes contain fusions between MGF110 proteins: ASFV-G contains fusions of MGF110 5L and 6L proteins, as well as 10L and 14L proteins

• ASFV-G ORF 13L has a frameshift mutation that splits the ORF into 13La and 13Lb genes

• Notably, MGF110-1L is present in all ASFV genomes, suggesting its essential role

Functional Differences:

• MGF 110-13L is highly antigenic and induces strong antibody responses in infected animals

• Studies on MGF 110-11L show that its deletion reduces pathogenicity while maintaining immunogenicity

• MGF 110-1L is uniquely present in all ASFV genomes, suggesting an essential function for viral survival

• Different MGF 110 proteins may have specialized roles in host immune evasion and virulence

Evolutionary Considerations:

• The presence of multiple diverse paralogs suggests gene duplication and divergent evolution

• Some MGF 110 genes show evidence of selective pressure, indicating roles in host adaptation

• Frameshift mutations and gene fusions point to ongoing evolutionary processes

• The conserved presence of MGF 110-1L across all ASFV genomes suggests a foundational role

This comparative analysis highlights the specialized nature of MGF 110-13L within the larger family, with particular importance in antigenic responses during infection. The family appears to have evolved from a common ancestor, with members acquiring specialized functions while maintaining core structural features related to membrane association.

How do the immunological properties of MGF 110-13L compare with other known ASFV antigenic proteins?

MGF 110-13L exhibits distinct immunological properties when compared to other ASFV antigenic proteins:

Comparative Antigenicity:

Experimental Comparison:
MGF 110-13L was specifically identified as antigenic when compared with six other transmembrane proteins (including EP153R and B475L), showing the strongest reaction signal with ASFV-infected pig serum . This indicates its superior immunogenicity among this group of proteins.

Epitope Characteristics:

• MGF 110-13L epitopes are linear and well-defined:

  • Epitope 8C3: 48WDCQDGICKNKITESRFIDS67

  • Epitope 10E4: 122GDHQQLSIKQ131

• Many other ASFV proteins contain conformational epitopes requiring intact protein structure

• The linear nature of MGF 110-13L epitopes makes them particularly suitable for peptide-based diagnostics

Temporal Expression and Immune Response:

• Different ASFV proteins are expressed at different stages of infection

• Early proteins (p30) elicit rapid antibody responses

• Structural proteins (p72) typically elicit strong but later responses

• The temporal dynamics of MGF 110-13L expression and corresponding antibody responses require further characterization

Potential for Multivalent Approaches:

• Combining MGF 110-13L epitopes with other antigenic proteins could enhance diagnostic sensitivity

• Multivalent vaccine approaches incorporating MGF 110-13L alongside other immunogenic proteins might provide broader protection

The distinct immunological properties of MGF 110-13L, particularly its strong antigenicity and highly conserved epitopes, position it as a valuable component for diagnostic development, especially when used in combination with other established ASFV antigenic markers.

What are the most promising future research directions for MGF 110-13L and its applications?

Based on current knowledge and identified gaps, several promising research directions for MGF 110-13L deserve priority:

1. Fundamental Structural and Functional Studies:

• Determine the three-dimensional structure of MGF 110-13L using X-ray crystallography or cryo-EM

• Characterize protein-protein interactions using comprehensive interactome approaches

• Investigate the role of glycosylation in protein function and immunogenicity

• Develop cell-based assays to elucidate the protein's role during different stages of viral infection

2. Advanced Diagnostic Applications:

• Develop and validate field-ready lateral flow assays using the 10E4 epitope

• Create multiplex diagnostic platforms incorporating MGF 110-13L alongside other ASFV antigens

• Establish serological testing algorithms optimized for different epidemiological scenarios

• Evaluate epitope-based assays in large-scale surveillance programs across diverse geographical regions

3. Vaccine Development Strategies:

• Generate and characterize MGF 110-13L deletion mutants as potential live-attenuated vaccine candidates

• Incorporate MGF 110-13L epitopes into subunit or vectored vaccine platforms

• Develop prime-boost regimens combining different vaccine approaches

• Evaluate cross-protection against diverse ASFV strains and genotypes

4. Integrated Systems Biology Approaches:

• Apply multi-omics technologies to understand the MGF 110-13L-host interaction network

• Develop computational models predicting functional impact of modifications to MGF 110-13L

• Utilize CRISPR-based screening to identify host factors interacting with MGF 110-13L

• Investigate the evolutionary relationships between MGF 110 family members across ASFV strains

5. Translational Research Priorities:

• Conduct field validation of MGF 110-13L-based diagnostics in endemic regions

• Evaluate MGF 110-13L-targeted interventions in controlled field trials

• Develop standardized reagents and reference materials for international harmonization

• Establish biobanking initiatives to support future research

Research Impact Projection:

The most immediate impact will likely come from diagnostic applications utilizing the already characterized epitopes, while vaccine development represents a longer-term but potentially higher-impact direction. Fundamental research into protein function remains essential to support all applied efforts.