Recombinant African swine fever virus Protein MGF 110-12L (Mal-017)

What is MGF 110-12L (Mal-017) and what is its role in African Swine Fever Virus?

MGF 110-12L (Mal-017) is a protein encoded by the multigene family 110 of African swine fever virus (ASFV). It belongs to a class of proteins that play significant roles in virus-host interactions and viral immune evasion strategies. The protein consists of 171 amino acids in its full-length form and is one of the several multigene family proteins that contribute to ASFV virulence and host range determination . As a component of the MGF 110 family, this protein participates in modulating host cell responses during infection, though its precise mechanisms remain under investigation by researchers worldwide.

How can I express and purify recombinant MGF 110-12L protein for research purposes?

For optimal expression and purification of MGF 110-12L (Mal-017), an E. coli expression system with His-tag modification has proven effective. The recommended protocol involves:

• Cloning the full-length sequence (amino acids 1-171) into an appropriate expression vector with a histidine tag

• Transforming the construct into an E. coli strain optimized for protein expression

• Inducing protein expression under controlled temperature and IPTG concentration

• Lysing cells and purifying the recombinant protein using nickel affinity chromatography

• Performing dialysis to remove imidazole and other contaminants

• Verifying protein integrity through SDS-PAGE and Western blotting

This approach yields functional recombinant protein suitable for downstream applications including antibody production, protein-protein interaction studies, and functional assays.

What expression systems are most suitable for producing MGF 110-12L for immunological studies?

• Baculovirus expression systems in insect cells, which provide eukaryotic processing capabilities

• Mammalian expression systems such as HEK293 or CHO cells for studies requiring authentic folding and modifications

• Cell-free expression systems for rapid screening of protein variants

The choice depends on the specific research objectives, with E. coli systems being preferred for structural studies and mammalian systems for functional immunological assays where conformational epitopes are important for antibody recognition.

What are the known functional domains within MGF 110-12L and how do they contribute to viral pathogenesis?

While the complete functional domain architecture of MGF 110-12L remains under investigation, research on ASFV multigene families suggests this protein contributes to viral pathogenesis through:

• Potential interference with host innate immune signaling pathways

• Possible modulation of host cell apoptotic responses

• Contribution to viral replication efficiency in specific host cell types

The MGF 110 family, including MGF 110-12L, has been implicated in host range determination and virulence, as evidenced by attenuation studies where deletion of these genes reduces viral pathogenicity . The specific functional domains responsible for these activities require further characterization through targeted mutagenesis and protein interaction studies.

How does MGF 110-12L interact with host cell proteins during ASFV infection?

• Yeast two-hybrid screening against porcine cellular protein libraries

• Co-immunoprecipitation followed by mass spectrometry analysis

• Proximity labeling approaches such as BioID or APEX in infected cells

• Surface plasmon resonance to confirm direct binding to candidate partners

Understanding these interactions would provide valuable insights into how MGF 110-12L contributes to viral replication and immune evasion strategies. Current research suggests that ASFV MGF proteins collectively function to antagonize host antiviral responses, but the specific partners of MGF 110-12L require dedicated investigation .

What methods are most effective for studying the subcellular localization of MGF 110-12L during infection?

To investigate the subcellular localization of MGF 110-12L during ASFV infection, researchers should consider:

• Immunofluorescence microscopy using specific antibodies against MGF 110-12L in infected cells

• Live-cell imaging of fluorescently tagged MGF 110-12L in ASFV-infected cells

• Subcellular fractionation followed by Western blot analysis

• Correlative light and electron microscopy for high-resolution localization studies

These approaches should be conducted at various time points post-infection to track potential changes in localization throughout the viral life cycle. Comparing localization in permissive versus non-permissive cells may also provide insights into functional relevance of this protein in different host contexts.

What experimental approaches should be used to assess the impact of MGF 110-12L on host immune responses?

To evaluate how MGF 110-12L influences host immune responses, researchers should implement:

• Comparative transcriptomics (RNA-seq) in cells infected with wild-type versus MGF 110-12L-deleted ASFV

• Proteomic analysis of signaling pathway activation in the presence/absence of MGF 110-12L

• ELISA and multiplex cytokine assays to measure inflammatory mediator production

• Flow cytometry to assess immune cell activation and phenotype changes

• In vivo immune response profiling in animals infected with parent or MGF 110-12L mutant viruses

These approaches would help determine whether MGF 110-12L modulates specific aspects of innate or adaptive immunity, such as type I interferon responses, inflammatory cytokine production, or antigen presentation pathways.

What is the current understanding of MGF 110-12L conservation across different ASFV isolates?

Although the search results don't provide specific information about MGF 110-12L conservation, addressing this question would involve:

• Comparative genomic analysis across sequenced ASFV isolates from different geographical regions and timeframes

• Phylogenetic analysis to determine evolutionary relationships and selection pressures

• Structural modeling to identify conserved functional domains versus variable regions

• Functional comparison of MGF 110-12L variants from different isolates

How has MGF 110-12L been utilized in the development of attenuated ASFV vaccine candidates?

The development of attenuated ASFV vaccine candidates has incorporated MGF 110-12L deletion as part of broader gene manipulation strategies:

• Multiple studies have created attenuated ASFV strains by deleting combinations of MGF genes, including those from MGF 110 family

• For example, deletion of six MGF genes (including MGF360-12L, MGF360-13L, MGF360-14L, MGF505-1R, MGF505-2R, and MGF505-3R) from ASFV HLJ/18 created an attenuated strain (HLJ/18–6GD) that provided 100% protection against challenge with wild-type virus

• Similar approaches with other ASFV isolates like Georgia 2007, Benin 97/1, and Malawi Lil-20/1 have demonstrated that MGF-deleted viruses can serve as effective live-attenuated vaccine candidates

While these studies typically delete multiple MGF genes simultaneously rather than MGF 110-12L alone, they demonstrate the importance of this gene family in virulence and the potential for targeting these genes in rational vaccine design.

What methodologies are recommended for evaluating immune responses to MGF 110-12L in vaccination studies?

For comprehensive evaluation of immune responses to MGF 110-12L in vaccination studies, researchers should implement:

• ELISA assays to measure antibody responses specific to MGF 110-12L

• ELISpot or intracellular cytokine staining to assess T-cell responses

• Neutralization assays to determine functional antibody activity

• Adoptive transfer experiments to evaluate the protective capacity of MGF 110-12L-specific immune responses

• Challenge studies comparing protection levels in animals immunized with different MGF 110-12L constructs or delivery systems

These approaches would help determine whether MGF 110-12L represents a viable target for subunit vaccine development or whether it primarily serves as a virulence factor that can be deleted to create attenuated live vaccines.

How does MGF 110-12L deletion compare with other gene modifications in ASFV vaccine development?

The comparative efficacy of different gene deletion strategies in ASFV vaccine development reveals important insights:

This data indicates that MGF gene deletions, including those affecting MGF 110 family members, produce attenuated viruses that can confer protection against challenge. Different deletion strategies show varying degrees of attenuation and protection, suggesting that optimal vaccine design may require precise combinations of gene modifications .

What are the challenges in studying MGF 110-12L protein-protein interactions and how can they be overcome?

Studying protein-protein interactions involving MGF 110-12L presents several challenges:

• Potential cytotoxicity when expressed in heterologous systems

• Conformational dependencies that may require viral context for proper folding

• Transient or weak interactions that are difficult to capture with conventional methods

• Limited availability of validated reagents specific to this protein

These challenges can be addressed through:

• Inducible expression systems with tight regulation to minimize toxicity

• Split-protein complementation assays to detect transient interactions in living cells

• Chemical crosslinking coupled with mass spectrometry for capturing weak interactions

• Development of high-affinity monoclonal antibodies against different epitopes of MGF 110-12L

• Computational prediction of interaction partners followed by targeted validation experiments

These approaches would enable more comprehensive characterization of the MGF 110-12L interactome and its functional significance in ASFV biology.

How can structural biology approaches enhance our understanding of MGF 110-12L function?

Structural biology techniques would significantly advance our understanding of MGF 110-12L by:

• X-ray crystallography or cryo-electron microscopy to determine three-dimensional structure

• NMR spectroscopy to analyze dynamic regions and conformational changes upon binding

• Hydrogen-deuterium exchange mass spectrometry to identify functional domains and binding interfaces

• Molecular dynamics simulations to predict functional motifs and interaction potential

• Structure-guided mutagenesis to validate functional predictions

These approaches would reveal the molecular architecture of MGF 110-12L and provide insights into its mode of action. Structural information would also guide rational design of inhibitors or modifications for vaccine development purposes.

What cutting-edge technologies could advance MGF 110-12L research in the context of host-pathogen interactions?

Several emerging technologies could significantly advance MGF 110-12L research:

• CRISPR-Cas9 genome editing of both viral and host genomes to investigate functional relationships

• Single-cell transcriptomics to analyze heterogeneity in host cell responses to MGF 110-12L

• Organoid models of porcine tissues to study MGF 110-12L function in physiologically relevant systems

• Proximity-dependent biotinylation (BioID or TurboID) to map the spatial interactome of MGF 110-12L

• Cryo-electron tomography to visualize MGF 110-12L in the context of viral replication complexes

• AlphaFold2 or similar AI-based structural prediction tools to model protein structures and interactions

Implementation of these technologies would provide unprecedented insights into the molecular mechanisms through which MGF 110-12L contributes to ASFV pathogenesis and host range determination, potentially revealing new targets for antiviral intervention or vaccine development.

What are common challenges in expressing and purifying functional MGF 110-12L and how can they be resolved?

Researchers working with MGF 110-12L may encounter several technical challenges:

• Poor solubility or protein aggregation during expression and purification

• Low yield of functional protein

• Insufficient purity for downstream applications

• Loss of biological activity during purification process

These issues can be addressed through:

• Optimization of expression conditions (temperature, induction time, media composition)

• Testing multiple solubility tags (MBP, GST, SUMO) beyond the standard His-tag

• Inclusion of stabilizing agents in purification buffers

• Implementation of multiple chromatography steps for increased purity

• Refolding protocols if inclusion body formation occurs

• Activity assays at each purification step to track functional protein recovery

Researchers should systematically optimize each parameter while maintaining focus on the intended application requirements for the purified protein.

How can I develop and validate antibodies against MGF 110-12L for research applications?

Developing reliable antibodies against MGF 110-12L requires:

• Careful epitope selection based on predicted antigenicity and accessibility

• Design of multiple immunization strategies (full-length protein, selected peptides, or fragments)

• Screening of antibodies against both recombinant protein and ASFV-infected cell lysates

• Validation through multiple approaches:

  • Western blotting under reducing and non-reducing conditions

  • Immunoprecipitation followed by mass spectrometry confirmation

  • Immunofluorescence microscopy with appropriate controls

  • Neutralization of known MGF 110-12L functions if applicable

• Characterization of cross-reactivity with related MGF proteins

• Determination of antibody performance in different applications (ELISA, flow cytometry, etc.)

This rigorous development and validation process ensures that resulting antibodies provide reliable tools for studying MGF 110-12L expression, localization, and interactions.

What experimental designs are most appropriate for studying the role of MGF 110-12L in ASFV immune evasion?

To effectively investigate MGF 110-12L's role in immune evasion, researchers should implement:

• Comparative studies between wild-type and MGF 110-12L-deleted viruses:

  • Measure type I interferon responses in infected macrophages

  • Analyze activation of pattern recognition receptors and downstream signaling

  • Evaluate antigen presentation and MHC expression

• Trans-complementation experiments:

  • Express MGF 110-12L in cells infected with deletion mutant virus

  • Determine which immune functions are restored by the protein alone

• Domain mapping studies:

  • Create truncated or point-mutated versions of MGF 110-12L

  • Identify regions responsible for specific immune evasion functions

• Temporal analysis:

  • Study immune responses at multiple time points post-infection

  • Determine when MGF 110-12L exerts its effects during viral replication cycle

These experimental approaches would provide a comprehensive understanding of how MGF 110-12L contributes to ASFV's ability to evade or manipulate host immune responses, informing both basic virology knowledge and applied vaccine development efforts.