Recombinant African swine fever virus Protein MGF 110-13L (War-020)

What is MGF 110-13L and what is its significance in ASFV research?

MGF 110-13L is an immunogenic protein encoded by African swine fever virus (ASFV). It has been identified as one of the antigenic proteins that induces strong antibody responses in infected pigs. The protein is significant in ASFV research because it shows strong reaction signals with ASFV-infected pig sera, making it valuable for both diagnostic development and understanding virus-host interactions. Recent studies have characterized it as a glycosylated homodimer when expressed in eukaryotic cells, with its outer-membrane domain containing 131 amino acids . The protein belongs to the multigene family 110 (MGF110), which is located in the Left Variable region of the ASFV genome .

What is the protein structure and post-translational modifications of MGF 110-13L?

MGF 110-13L is expressed as a partially glycosylated homodimer in eukaryotic expression systems. When analyzed by SDS-PAGE, the purified recombinant MGF 110-13L protein exhibits two main bands near or above 15 kDa. After treatment with PNGase F (which removes N-glycans), the protein appears as a single band of approximately 15 kDa, confirming glycosylation. The protein contains a predicted N-glycosylation site (68NHS70) in its outer-membrane domain. Under non-reducing conditions (absence of β-mercaptoethanol), the protein shows three bands near or above 35 kDa, which likely represent different glycosylation states of the homodimer: glycosylated-glycosylated, glycosylated-unglycosylated, and unglycosylated-unglycosylated forms .

How conserved is MGF 110-13L across different ASFV isolates?

The MGF 110-13L protein shows high conservation across ASFV isolates. Analysis of 191 MGF 110-13L protein sequences in the NCBI database (as of May 2024) revealed 59 full-length sequences. Sequence alignment showed that both identified epitopes (48WDCQDGICKNKITESRFIDS67 and 122GDHQQLSIKQ131) are 100% conserved (59/59) among these sequences. This high conservation suggests that MGF 110-13L likely serves an important function in the virus life cycle, despite not being essential for virulence as shown with other MGF family members .

What are the recommended methods for expressing recombinant MGF 110-13L?

For expressing MGF 110-13L, two primary systems have been documented in research:

• Mammalian expression system: Using BHK-21 cells transfected with plasmids containing the MGF 110-13L ectodomain (amino acids 1-131). This approach yields partially glycosylated protein that forms homodimers, closely resembling the natural state of the protein. The protocol involves:

  • Linearizing expression plasmids with SspI

  • Transfection of BHK-21 cells

  • Selection using G418

  • Cloning and screening of stable cell lines by IFA and Western blot

• E. coli expression system: For non-glycosylated protein production, particularly for applications where glycosylation is not critical. This method is more cost-effective but lacks post-translational modifications .

The choice between these systems depends on the research question, with the mammalian system preferred for structural and functional studies requiring native-like protein.

What purification strategies are effective for MGF 110-13L protein?

For purification of MGF 110-13L, the following method has been demonstrated to be effective:

• Affinity chromatography using Ni-NTA: For His-tagged recombinant MGF 110-13L, affinity chromatography with an Ni-NTA agarose column can be used to purify the protein from cell culture supernatants.

• Storage conditions: After purification, the protein should be stored at -80°C until use. For commercial preparations, storage in Tris-based buffer with 50% glycerol at -20°C is recommended, with working aliquots stored at 4°C for up to one week to avoid repeated freeze-thaw cycles .

The protein concentration can be determined using a BCA protein assay kit. When analyzing the purified protein, researchers should be aware of the multiple bands that appear on SDS-PAGE due to glycosylation and dimerization.

What epitopes have been identified on MGF 110-13L and how were they mapped?

Two linear epitopes have been identified on the MGF 110-13L protein using monoclonal antibodies (mAbs):

• First epitope (mAb 8C3): 48WDCQDGICKNKITESRFIDS67

• Second epitope (mAb 10E4): 122GDHQQLSIKQ131

These epitopes were mapped using the following methodology:

• Generation of mAbs by immunizing BALB/c mice with purified MGF 110-13L protein

• Design of eight partially overlapping short peptides (P1-P8) covering the entire length of the MGF 110-13L outer-membrane domain

• Expression of these peptides as fusion proteins with maltose-binding protein (MBP)

• Screening of peptides using mAbs by ELISA and Western blot

• Further fine-mapping using truncated peptide fusion proteins to identify the core sequences

Notably, when tested with sera from ASFV-infected pigs, peptide EP8 (containing the second epitope) produced strong reaction signals, indicating it is an antigenic epitope recognized during natural infection .

How can we generate and characterize monoclonal antibodies against MGF 110-13L?

The generation and characterization of monoclonal antibodies against MGF 110-13L can be achieved using the following protocol:

• Immunization:

  • Immunize BALB/c mice subcutaneously with purified protein emulsified with Freund's adjuvant

  • Administer three immunizations at 21-day intervals

  • Give a final booster by intraperitoneal injection with soluble protein alone 3 days before cell fusion

• Hybridoma generation:

  • Fuse mouse splenocytes with SP2/0 cells

  • Screen hybridomas by ELISA

  • Subclone positive cell lines by limiting dilution

• Antibody purification and characterization:

  • Purify monoclonal antibodies using affinity chromatography with a Protein G column

  • Identify heavy and light chains using an antibody isotyping kit

  • Determine antibody titers by ELISA

  • Verify specificity by Western blot against MGF 110-13L in lysates of ASFV-infected cells

In one study, this methodology yielded two hybridomas (8C3 and 10E4) that produced mAbs with titers above 51,200 and 102,400, respectively, at a concentration of 1 mg/mL .

How can MGF 110-13L be utilized for ASFV serodiagnosis?

MGF 110-13L shows promise as a diagnostic antigen for ASFV antibody detection, particularly through the use of its epitope peptides. The methodology for developing a serodiagnostic test includes:

• Epitope-based dot blot assay:

  • Synthesize peptides corresponding to identified epitopes (particularly the 122GDHQQLSIKQ131 epitope)

  • Apply peptides to membrane for dot blot assay

  • Test with sera from experimentally and naturally infected pigs

  • Evaluate sensitivity and specificity

Research has shown that peptide EP8 (containing the 10E4 epitope) reacted positively with all tested ASFV-infected pig sera, including five experimentally infected and five naturally infected samples. This suggests that epitope-based diagnostic assays could be developed as alternatives to traditional whole-virus antigen tests .

What is the relationship between MGF 110-13L and other MGF family proteins in ASFV?

MGF 110-13L belongs to the MGF110 family of genes located in the Left Variable region of the ASFV genome. While specific comparative data between MGF 110-13L and other MGF proteins is limited, research on the MGF family provides context:

• Genomic organization: The MGF110 family in ASFV Georgia strain (ASFV-G) spans approximately 9kb between nucleotide positions 7004 and 16031, containing all MGF110 genes and one MGF100-1R gene .

• Functional distinctions:

  • MGF 110-13L has been identified as strongly antigenic

  • MGF 110-1L appears to be non-essential for virulence, as its deletion does not significantly affect virus replication or clinical disease progression

  • MGF 110-9L shows reduced ability to replicate in vitro in primary swine macrophage cell cultures

  • MGF 110-5L-6L deletion does not impact virulence or virus replication

• Role in immune modulation: Other MGF family members (particularly MGF360 and MGF505) are known to modulate interferon responses and host immune function. For example, MGF360-9L is involved in down-regulation of interferon expression .

These differences suggest specialized functions for different MGF family members in virus-host interactions, with MGF 110-13L likely playing a role in antigenicity and possibly structural functions.

How does post-translational modification of MGF 110-13L affect its immunogenicity and function?

The impact of glycosylation on MGF 110-13L's immunogenicity and function represents an important research question. While direct comparative studies are not fully documented, the following methodological approach could address this question:

• Comparative immunogenicity analysis:

  • Express MGF 110-13L in both mammalian systems (producing glycosylated protein) and bacterial systems (producing non-glycosylated protein)

  • Immunize separate groups of animals with each protein preparation

  • Compare antibody titers, specificity, and neutralizing capacity

  • Analyze T-cell responses to determine if glycosylation affects T-cell epitope presentation

• Structural impact assessment:

  • Compare the stability and solubility of glycosylated versus non-glycosylated forms

  • Perform circular dichroism or other structural analyses to detect conformational differences

  • Evaluate whether glycosylation affects dimerization capability

Current evidence suggests that MGF 110-13L contains an N-glycosylation site (68NHS70) and forms homodimers in its native state. The observation that purified protein from mammalian cells exhibits both glycosylated and non-glycosylated forms indicates that glycosylation may not be essential for basic protein folding but could influence other functional aspects .

What role might MGF 110-13L play in ASFV pathogenesis compared to other MGF family proteins?

Understanding MGF 110-13L's role in pathogenesis requires comparative analysis with other MGF proteins and deletion studies:

• Methodological approach for functional analysis:

  • Generate deletion mutants of ASFV lacking MGF 110-13L (similar to studies done with MGF 110-1L)

  • Evaluate replication kinetics in primary swine macrophage cell cultures

  • Compare virulence in experimental swine infections

  • Analyze transcriptome/proteome changes in infected cells with and without MGF 110-13L

• Comparative framework:

  MGF Protein	Effect of Deletion	Role in Immune Modulation	Conservation
  MGF 110-13L	Not fully determined	Identified as immunogenic	Highly conserved (100% for key epitopes)
  MGF 110-1L	Non-essential for virulence	Not determined	Conserved
  MGF 110-9L	Reduced replication in vitro	Not determined	Not reported
  MGF360/505 (various)	Attenuation of virulence	Modulation of IFN response	Variable

While MGF 110-13L has been identified as highly antigenic, its direct role in virulence is not yet established. Researchers should note that while some MGF family members (like those in MGF360/505) clearly contribute to virulence by suppressing host immune responses, others may have structural roles or functions in viral replication and assembly .

How can structural biology techniques be applied to better understand MGF 110-13L's function and interactions?

Structural characterization of MGF 110-13L would significantly advance understanding of its function. A comprehensive approach would include:

• Structural determination methodology:

  • X-ray crystallography of the purified protein (both glycosylated and non-glycosylated forms)

  • Cryo-electron microscopy to visualize the protein in different contexts

  • NMR spectroscopy for dynamic structural analysis

  • In silico molecular modeling based on the amino acid sequence

• Protein-protein interaction studies:

  • Yeast two-hybrid or pull-down assays to identify viral or host protein partners

  • Surface plasmon resonance to quantify binding affinities

  • Proximity labeling in infected cells to identify in vivo interactions

• Structure-function analysis:

  • Site-directed mutagenesis of key residues, particularly those in identified epitopes

  • Functional assays to determine the effect of mutations on protein activity

  • Analysis of how dimerization affects function using mutations that disrupt this property

These approaches would help determine if MGF 110-13L serves primarily as a structural component of the virus, participates in host immune evasion, or has enzymatic activity. The transmembrane nature of the protein suggests it could be involved in virus assembly, entry, or membrane modification during infection .

What potential does MGF 110-13L have for ASFV vaccine development?

MGF 110-13L shows promise for ASFV vaccine development based on its immunogenic properties. A methodological framework for exploring this potential includes:

• Subunit vaccine approach:

  • Express and purify recombinant MGF 110-13L using appropriate systems

  • Formulate with adjuvants suitable for swine vaccination

  • Evaluate antibody responses, T-cell activation, and protection against challenge

  • Compare whole protein versus epitope-based vaccines

• Inclusion in multi-antigen formulations:

  • Combine MGF 110-13L with other immunogenic ASFV proteins (p72, p54, p30, CD2v)

  • Assess potential synergistic protection

  • Evaluate cross-protection against different ASFV isolates

Current research on other MGF family proteins indicates that deletion of certain MGF genes (particularly in the MGF360/505 families) has resulted in attenuated viruses that can confer protection against challenge. While MGF 110-13L's role in virulence is not established, its high conservation and strong immunogenicity make it a candidate for inclusion in subunit vaccine formulations or as a target for rationally designed live attenuated vaccines .

How might systematic genetic analysis of MGF 110-13L variants contribute to understanding ASFV evolution and host adaptation?

Systematic analysis of MGF 110-13L across ASFV variants could provide insights into virus evolution and host adaptation through the following methods:

• Comparative genomic analysis:

  • Sequence MGF 110-13L from diverse ASFV isolates (domestic pig isolates, warthog isolates, different geographical regions)

  • Perform phylogenetic analysis to trace evolutionary relationships

  • Identify selection pressures using dN/dS ratios

  • Map variations to functional domains and epitopes

• Host adaptation studies:

  • Compare MGF 110-13L sequences from ASFV isolates adapted to different hosts (domestic pigs vs. wild suids)

  • Identify amino acid changes that correlate with host switches

  • Functionally test these variants in vitro and in vivo

• Cross-species binding assays:

  • Express MGF 110-13L variants from different isolates

  • Test binding to cells or receptors from different host species

  • Identify regions critical for host-specific interactions

This approach would help determine if MGF 110-13L contributes to the different outcomes of ASFV infection observed in domestic pigs (typically fatal) versus African wild suids (typically asymptomatic). Current data shows high conservation of key epitopes, but comprehensive analysis across more diverse isolates is needed .