Recombinant African swine fever virus Protein MGF 110-9L (Pret-021)

What is MGF 110-9L (Pret-021) and what is its significance in ASFV research?

MGF 110-9L (Pret-021) is a protein belonging to the multigene family 110 (MGF-110) of African swine fever virus (ASFV). It is encoded by a gene approximately 873 nucleotides in length in genotype II ASFV. The protein is positioned on the reverse strand between nucleotide positions 11627 and 12499 of the ASFV CN/GS/2018 genome. MGF 110-9L is particularly significant in ASFV research because deletion studies have demonstrated its role in viral replication and virulence. The MGF-110 family contains proteins with a hydrophobic NH2-terminal sequence and a conserved cysteine-rich domain, with potential roles in host range determination, viral virulence, and preparing the endoplasmic reticulum for viral morphogenesis .

Recent research has shown that deletion mutants lacking the MGF 110-9L gene demonstrate attenuated virulence in pigs, suggesting this protein's importance in ASFV pathogenesis and its potential application in vaccine development strategies .

How conserved is MGF 110-9L among different ASFV isolates?

Analysis of MGF 110-9L protein sequences from various ASFV isolates reveals a high degree of conservation, particularly among genotype II ASFV strains. Comparative studies examining 10 different African, European, and Caribbean pathogenic virus isolates demonstrated that the 290-amino acid MGF 110-9L protein is detected in most isolates, with only minor variations observed .

Some isolates feature a truncated C-terminus of the sequence, but the core functional domains remain largely conserved. This high level of conservation suggests that MGF 110-9L serves an important function in the ASFV life cycle that has been maintained through evolutionary pressure .

The conservation pattern of MGF 110-9L contrasts with some other ASFV proteins that show greater variability across isolates, highlighting its potential fundamental role in viral biology.

When is MGF 110-9L expressed during the ASFV replication cycle?

MGF 110-9L is transcribed early in the ASFV replication cycle. Time-course experiments tracking the expression of MGF 110-9L at the mRNA level revealed detectable expression as early as 3 hours post-infection (h.p.i.) .

The expression pattern of MGF 110-9L closely resembles that of the p30 protein, a well-established early viral protein, rather than the late-expressed p72 protein (which typically appears around 10 h.p.i.). This temporal expression pattern provides important insights into the potential role of MGF 110-9L in the viral replication cycle .

The early expression timing suggests MGF 110-9L may be involved in initial phases of viral infection such as host immune evasion, replication complex formation, or early modulation of host cell processes .

What experimental systems are available for studying MGF 110-9L function?

Several experimental systems are available for investigating MGF 110-9L function:

• Primary Swine Macrophage Cultures: Primary alveolar macrophages (PAMs) are the primary cell type for studying ASFV proteins in vitro. These cells support ASFV replication and can be used for viral growth kinetics, protein expression, and functional studies of MGF 110-9L .

• Recombinant Protein Expression Systems: E. coli-based expression systems have been successfully used to produce recombinant MGF 110-9L protein with His-tags for purification and functional studies .

• CRISPR-Cas9 Gene Editing: This approach has been used to generate MGF 110-9L deletion mutants (ASFV-Δ9L) from virulent ASFV strains, enabling comparative studies between wild-type and mutant viruses .

• Animal Models: Landrace-crossed pigs have been employed for in vivo studies to assess the effects of MGF 110-9L deletion on virulence, clinical symptoms, viremia, and immune responses .

• Fluorescence Microscopy: Using GFP-tagged viruses (replacing the MGF 110-9L gene with eGFP) allows for visualization of viral infection processes and quantification via fluorescence .

These experimental systems provide complementary approaches for comprehensive analysis of MGF 110-9L's roles in ASFV biology.

How does deletion of MGF 110-9L affect ASFV replication in vitro and virulence in vivo?

The deletion of MGF 110-9L results in significant changes to both in vitro replication and in vivo virulence:

In vitro effects:

• ASFV-Δ9L (MGF 110-9L deletion mutant) displays significantly slower growth kinetics in primary swine macrophages compared to parental ASFV strains

• Virus yields in ASFV-Δ9L are consistently 5-10 fold lower than the parental virus across multiple time points (12, 24, 36, and 48 h.p.i.)

• The deletion significantly hinders the virus's ability to replicate in primary swine macrophage cell cultures

In vivo effects:

• Pigs inoculated intramuscularly with a low dose (10 HAD50) of ASFV-Δ9L generally remained clinically normal during a 21-day observation period

• Three of five ASFV-Δ9L-infected animals showed reduced clinical manifestations with:

  • Low viremia titers

  • Low virus shedding

  • Development of strong virus-specific antibody responses

• This indicates partial attenuation of the ASFV-Δ9L strain in pigs

These findings suggest that MGF 110-9L is an important virulence factor that contributes significantly to ASFV replication efficiency and pathogenesis, making it a potential target for attenuated vaccine development.

What methodologies can be used to assess the interaction of MGF 110-9L with host factors?

Several advanced methodologies can be employed to investigate MGF 110-9L interactions with host factors:

• Co-immunoprecipitation (Co-IP): Using antibodies against MGF 110-9L or potential host interaction partners to pull down protein complexes from infected cells, followed by western blotting or mass spectrometry to identify binding partners.

• Yeast Two-Hybrid (Y2H) Screening: Expressing MGF 110-9L as bait against a porcine macrophage cDNA library to identify potential protein-protein interactions.

• Proximity Ligation Assay (PLA): For visualization of protein interactions in situ within infected cells, providing spatial context for interactions.

• BioID or APEX Proximity Labeling: Fusing MGF 110-9L to biotin ligase to biotinylate proteins in close proximity, followed by streptavidin pull-down and mass spectrometry.

• Chromatin Immunoprecipitation (ChIP): If MGF 110-9L potentially interacts with host chromatin or DNA-binding proteins, ChIP can identify these associations.

• Confocal Microscopy with Co-localization Analysis: Using fluorescently tagged MGF 110-9L to observe co-localization with host cellular structures or proteins.

• Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC): For determining binding kinetics and thermodynamics between purified recombinant MGF 110-9L and candidate host proteins.

These methodologies should ideally be used in combination to validate interactions from multiple angles and understand the functional significance of MGF 110-9L-host factor interactions in ASFV pathogenesis.

How can recombinant MGF 110-9L protein be used in immunological studies and vaccine development?

Recombinant MGF 110-9L protein offers several applications in immunological studies and vaccine development:

• Antibody Production: Purified recombinant MGF 110-9L can be used to generate specific polyclonal or monoclonal antibodies for diagnostic assays, immunohistochemistry, and tracking viral infection .

• ELISA Development: The recombinant protein can serve as an antigen in ELISA-based diagnostic tests to detect anti-ASFV antibodies in pig serum, contributing to surveillance programs .

• Epitope Mapping: Systematic analysis of MGF 110-9L can identify immunodominant epitopes that might be incorporated into subunit or epitope-based vaccines.

• Immunogenicity Assessment: Evaluating the ability of recombinant MGF 110-9L to stimulate immune responses in vitro using peripheral blood mononuclear cells or in animal models.

• Correlates of Protection Studies: Comparing antibody responses against MGF 110-9L in animals that survive ASFV-Δ9L infection versus those that succumb to wild-type infection can help identify protective immune signatures .

• Subunit Vaccine Candidates: The protein could be incorporated into multicomponent subunit vaccines alongside other ASFV immunogens.

• Adjuvant Formulation Testing: Testing various adjuvant combinations with recombinant MGF 110-9L to enhance immunogenicity and protective efficacy.

The finding that pigs infected with ASFV-Δ9L develop strong ASFV-specific antibody responses, including detectable IgG and IgM by 5-11 days post-inoculation, suggests that properly presented MGF 110-9L might contribute to protective immunity against ASFV challenge .

What are the challenges in studying MGF 110-9L's contribution to ASFV pathogenesis?

Researchers face several significant challenges when investigating MGF 110-9L's role in ASFV pathogenesis:

• Biosafety Concerns: ASFV is a high-consequence pathogen requiring specialized BSL-3 facilities, limiting the number of laboratories able to conduct live virus studies.

• Functional Redundancy: The MGF-110 family contains multiple members that may have overlapping functions, making it difficult to attribute specific phenotypes solely to MGF 110-9L deletion.

• Limited Natural Host Range: ASFV primarily infects domestic and wild pigs, requiring specialized animal facilities and ethical considerations for in vivo studies.

• Cellular Tropism: ASFV predominantly replicates in macrophages, which can be challenging to maintain in culture and manipulate genetically.

• Technical Hurdles: Generation of gene-deleted ASFV mutants like ASFV-Δ9L requires sophisticated genome editing techniques and multiple rounds of purification (11 rounds reported in the literature) .

• Complex Virus-Host Interactions: MGF 110-9L likely participates in multiple virus-host interactions across different stages of infection, making comprehensive characterization difficult.

• Incomplete Understanding of Function: Despite knowing that MGF 110-9L is involved in virulence, its precise biochemical and molecular functions remain largely uncharacterized.

• Variability in Host Response: Individual pigs may respond differently to infection with wild-type or mutant ASFV strains, requiring larger sample sizes for statistically significant results.

Addressing these challenges requires multidisciplinary approaches combining virology, immunology, genomics, and advanced imaging techniques to fully elucidate MGF 110-9L's role in ASFV biology.

How does MGF 110-9L compare functionally to other members of the MGF-110 family and other ASFV virulence factors?

MGF 110-9L shares structural similarities with other MGF-110 family members but appears to have distinct functional roles in ASFV biology:

Comparison within MGF-110 family:

• All MGF-110 proteins contain hydrophobic NH2-terminal sequences and conserved cysteine-rich domains

• Unlike some other MGF-110 members, MGF 110-9L appears to be particularly important for efficient viral replication in macrophages

• While some MGF-110 family proteins may be involved in viral morphogenesis via endoplasmic reticulum modification, MGF 110-9L's deletion primarily affects replication efficiency and virulence

Comparison with other ASFV virulence factors:

• MGF 110-9L appears to function early in infection (similar to p30), while other virulence factors like MGF-360-10L (which targets JAK1 for degradation) may have more specific immunomodulatory roles

• Unlike some ASFV proteins that directly antagonize specific host immune pathways, MGF 110-9L appears to have broader effects on viral replication

• The partial attenuation observed with MGF 110-9L deletion is less dramatic than deletion of some other ASFV virulence genes, suggesting it contributes to but is not solely responsible for virulence

Functional hierarchy in pathogenesis:

Understanding these functional differences and similarities is crucial for developing comprehensive models of ASFV pathogenesis and identifying optimal targets for intervention strategies.

What are the optimal conditions for working with recombinant MGF 110-9L protein?

Based on available information about recombinant MGF 110-9L protein, the following methodological considerations should be observed:

• Storage Conditions:

  • Store at -20°C/-80°C upon receipt

  • Aliquot the protein to avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

  • Long-term storage benefits from the addition of glycerol (recommended 50% final concentration)

• Reconstitution Protocol:

  • Briefly centrifuge vials prior to opening to bring contents to the bottom

  • Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • For optimal stability, add glycerol to 5-50% final concentration

• Buffer Considerations:

  • The protein is typically supplied in Tris/PBS-based buffer with 6% Trehalose, pH 8.0

  • For functional studies, buffer compatibility should be tested beforehand

• Application-Specific Considerations:

  • For SDS-PAGE applications, protein purity is typically greater than 90%

  • For immunological assays, consider potential effects of the His-tag on epitope accessibility

  • For structural studies, the tag may need to be removed using appropriate proteases

• Quality Control Metrics:

  • Verify protein integrity by SDS-PAGE before experimental use

  • Consider Western blot confirmation of identity using anti-His antibodies or ASFV-specific antibodies

These methodological considerations help ensure reliable and reproducible results when working with recombinant MGF 110-9L protein in research applications.

What protocols are recommended for generating and validating MGF 110-9L deletion mutants?

The generation and validation of MGF 110-9L deletion mutants require rigorous methodological approaches:

Generation Protocol:

• CRISPR-Cas9 Gene Editing System:

  • Design guide RNAs targeting sequences flanking the MGF 110-9L gene

  • Create a donor template containing a reporter gene (e.g., eGFP under the control of the ASFV p72 promoter) flanked by homology arms

  • Transfect primary macrophages with CRISPR-Cas9 components and donor template, followed by ASFV infection

• Viral Purification:

  • Multiple rounds of limiting dilution and plaque purification are necessary (up to 11 rounds reported)

  • Selection based on fluorescence if using a fluorescent reporter gene replacement

  • Single-plaque isolation to ensure genetic homogeneity

Validation Methods:

• Genomic Verification:

  • PCR amplification using primers flanking the deletion site

  • Whole-genome sequencing to confirm the deletion and absence of unintended mutations

  • Restriction enzyme digestion analysis of PCR products

• Functional Validation:

  • Growth curve analysis in primary macrophages comparing deletion mutant to parental virus

  • Quantification using HAD50 or TCID50 assays

  • Fluorescence microscopy to confirm reporter gene expression

• In Vivo Characterization:

  • Intramuscular inoculation of pigs with standardized viral doses

  • Daily monitoring of temperature and clinical signs

  • Sample collection for viremia quantification and antibody response assessment

  • Comparative analysis with parental virus to establish attenuation phenotype

Following these rigorous protocols ensures the generation of well-characterized MGF 110-9L deletion mutants suitable for mechanistic studies and potential vaccine development.

What are the most promising future research avenues for understanding MGF 110-9L function?

Several promising research directions could advance our understanding of MGF 110-9L function:

• Structural Biology Approaches:

  • X-ray crystallography or cryo-EM studies of MGF 110-9L to determine its three-dimensional structure

  • Structural comparisons with other viral proteins to identify functional motifs

  • Protein-protein docking simulations to predict potential host interactions

• Systems Biology Integration:

  • Transcriptomic and proteomic profiling comparing wild-type and MGF 110-9L-deleted ASFV infection

  • Network analysis to position MGF 110-9L within viral-host interaction networks

  • Temporal multi-omics to understand dynamic changes during infection

• Host Range Determinants:

  • Investigation of MGF 110-9L's potential role in tick-pig transmission cycles

  • Comparative studies across different host species' macrophages to identify host-specific interactions

• Immune Evasion Mechanisms:

  • Detailed characterization of how MGF 110-9L might interfere with innate immune sensing pathways

  • Investigation of potential effects on interferon signaling or inflammasome activation

• Rational Vaccine Design:

  • Structure-guided modifications to enhance immunogenicity while removing virulence functions

  • Combinatorial deletion approaches targeting MGF 110-9L alongside other virulence factors

  • Development of DIVA (Differentiating Infected from Vaccinated Animals) strategies incorporating MGF 110-9L

• Advanced Imaging Studies:

  • Live-cell imaging using fluorescently tagged MGF 110-9L to track its localization during infection

  • Super-resolution microscopy to visualize interactions with host cellular structures

These research directions would significantly advance our understanding of MGF 110-9L's role in ASFV biology and potentially lead to novel intervention strategies against this economically devastating disease.

How might MGF 110-9L research contribute to ASFV vaccine development efforts?

The research on MGF 110-9L offers several promising avenues for advancing ASFV vaccine development:

• Live-Attenuated Vaccine Platforms:

  • The partial attenuation observed in ASFV-Δ9L mutants suggests this deletion could be incorporated into rationally designed live-attenuated vaccine candidates

  • Combining MGF 110-9L deletion with other attenuating mutations could achieve an optimal balance of safety and immunogenicity

• Immune Correlates of Protection:

  • Studying the antibody and cellular immune responses in pigs that survive ASFV-Δ9L infection can identify immunological signatures associated with protection

  • The gradual increase in p30-specific antibodies and detectable IgG/IgM responses observed in surviving pigs provides valuable insights

• Vectored Vaccine Approaches:

  • MGF 110-9L could be expressed in viral vectors (e.g., adenovirus or vaccinia virus) as part of multi-antigen ASFV vaccine candidates

  • The early expression of MGF 110-9L during infection suggests it might be an important target for early immune responses

• DIVA Strategies:

  • MGF 110-9L deletion mutants could form the basis for vaccines that allow Differentiation of Infected from Vaccinated Animals

  • Diagnostic tests targeting MGF 110-9L could help distinguish vaccine-induced immunity from natural infection

• Adjuvant Development:

  • Understanding how MGF 110-9L interacts with the host immune system could inform the design of adjuvants that specifically enhance protective responses against ASFV

• Cross-Protection Assessment:

  • Evaluating the protection conferred by MGF 110-9L-based vaccines against heterologous ASFV challenges would be critical given the high conservation of this protein across isolates

The findings that ASFV-Δ9L induces significant antibody responses while demonstrating attenuated virulence make this a particularly promising direction for vaccine research efforts against a disease for which no commercial vaccine is currently available .

What are the key takeaways about MGF 110-9L for researchers new to the field?

Researchers entering the field of ASFV MGF 110-9L studies should consider these essential takeaways:

• Fundamental Importance: MGF 110-9L is a highly conserved 290-amino acid protein in the ASFV MGF-110 family that plays a significant role in viral replication efficiency and virulence.

• Expression Pattern: The protein is expressed early in infection (similar to p30), suggesting involvement in initial infection processes rather than late assembly or maturation events.

• Genetic Manipulation: Deletion of the MGF 110-9L gene results in attenuated virulence in pigs and reduced replication capacity in primary macrophages, highlighting its potential as a target for vaccine development.

• Immunological Relevance: Pigs infected with MGF 110-9L deletion mutants develop strong virus-specific antibody responses, including detectable IgG and IgM, suggesting the protein's deletion may enhance immunogenicity.

• Experimental Accessibility: Recombinant forms of MGF 110-9L are available for research purposes, enabling a wide range of biochemical, immunological, and structural studies outside of high-containment facilities.

• Knowledge Gaps: Despite its importance, the precise molecular mechanisms by which MGF 110-9L contributes to viral replication and virulence remain incompletely understood, offering numerous opportunities for novel research.

• Interdisciplinary Approaches: The most successful research on MGF 110-9L will likely combine virology, immunology, structural biology, and systems biology approaches to fully elucidate its functions.