Recombinant African swine fever virus Protein MGF 110-1L (War-006)

What is the MGF 110-1L protein and why is it significant in ASFV research?

MGF 110-1L is a protein belonging to the multi-gene family 110 (MGF110) of African swine fever virus. It holds particular significance as the only MGF110 family member that is present in all sequenced ASFV isolates, suggesting a potentially critical role in viral survival or function . The protein is typically 196-271 amino acids in length, with size variations primarily due to C-terminal extensions in some isolates . MGF 110-1L is transcribed as an early viral gene during infection, following a transcriptional pattern similar to other early proteins like p30 (CP204L) . Despite its universal conservation across ASFV strains, experimental deletion has demonstrated that it is non-essential for viral replication in vitro and does not significantly affect virulence in vivo, presenting an interesting paradox for researchers .

How is MGF 110-1L positioned within the ASFV genome?

The MGF 110-1L gene is strategically located in the Left Variable region of the ASFV genome. In the Georgia 2007 isolate (ASFV-G), it is positioned between nucleotide positions 7004 and 7648 . This region contains all MGF110 family genes and one MGF100 gene (MGF100-1R). The entire MGF110 family spans approximately 9kb between positions 7004 and 16031 in ASFV-G . This genomic positioning in the variable region is significant as it suggests potential involvement in host adaptation and immune evasion, which are commonly associated with genes in variable regions of viral genomes.

What is known about MGF 110-1L expression during viral infection?

Transcriptional analysis using microarray data has revealed that MGF 110-1L, like other MGF110 family genes, is expressed at all time points during ASFV infection. The expression pattern shows an initial decrease from 3 to 9 hours post-infection (hpi), followed by increased expression from 12 to 18 hpi . This pattern mirrors that of established early proteins such as p30, indicating that MGF 110-1L functions as an early viral protein in the replication cycle . The timing of expression suggests a potential role in establishing infection or modulating early host responses, though further proteomic studies would be beneficial to confirm protein-level dynamics during infection.

How conserved is the MGF 110-1L gene across different ASFV isolates?

MGF 110-1L demonstrates remarkable conservation across ASFV isolates. Multiple sequence alignment analyses reveal that while the protein varies in length from 196 to 271 amino acids across different isolates, the sequence itself is highly conserved within comparable length variants . Genotype II isolates, including the Georgia 2007 strain, show 100% amino acid identity for the first 196 amino acids of the protein . Even across different genotypes, approximately 84% of residues in the ASFV-G protein sequence are conserved . This high degree of conservation, particularly within genotypes, strongly suggests functional importance and potential selective pressure to maintain the protein structure.

What is the structural organization of the MGF 110-1L gene in ASFV-G?

In the Georgia 2007 isolate (ASFV-G), MGF 110-1L encodes a protein of 214 amino acids. The gene spans 645 nucleotides from position 7004 to 7648 in the viral genome . Research has shown that this gene can be completely deleted and replaced with marker genes (such as mCherry under the p72 promoter) using homologous recombination techniques . The successful generation of deletion mutants indicates that the flanking regions (positions 6003-7003 and 7649-8649) provide sufficient homology for targeted genetic manipulation of this locus .

How does MGF 110-1L compare with other members of the MGF110 gene family?

While MGF 110-1L is universally conserved across all ASFV isolates, other MGF110 family members show considerable variation in presence and structure between different viral strains. The ASFV-G genome contains 11 different MGF110 family genes (1L, 2L, 3L, 4L, 5L-6L, 7L, 9L, 10-14L, 12L, 13La, and 13Lb) . Some MGF110 genes exhibit fusion events or frameshift mutations; for example, ASFV-G contains fusions between MGF110 5L and 6L proteins, as well as between 10L and 14L proteins, while the ORF 13L has a frameshift mutation splitting it into 13La and 13Lb genes . Unlike MGF 110-1L, other family members are not consistently present across all isolates, suggesting they may have more specialized or accessory functions in viral biology.

How can researchers generate MGF 110-1L deletion mutants for functional studies?

Generating MGF 110-1L deletion mutants involves a systematic process of homologous recombination. A detailed methodological approach includes:

• Construction of recombination transfer vector: Researchers should design a vector (like p72mCherryΔMGF110-1L) containing:

  • Left arm homology region (e.g., positions 6003-7003)

  • Right arm homology region (e.g., positions 7649-8649)

  • Reporter gene cassette (e.g., mCherry under p72 promoter)

• Transfection and homologous recombination:

  • Infect macrophage cultures with parental virus (e.g., ASFV-G)

  • Transfect infected cells with the recombination transfer vector

  • Allow homologous recombination to occur, resulting in replacement of the target gene with the reporter cassette

• Purification of recombinant virus:

  • Conduct successive rounds of limiting dilution purification

  • Select wells containing the highest dilution with detectable reporter expression (e.g., mCherry fluorescence)

  • Verify purity through multiple passages

• Verification of deletion mutant:

  • Extract viral DNA and perform next-generation sequencing

  • Confirm complete deletion of the target gene and proper insertion of the reporter cassette

  • Verify genome integrity to ensure no additional mutations occurred during recombination

This methodology has been successfully employed to create ASFV-G-ΔMGF110-1L, demonstrating the feasibility of this approach for functional studies of MGF 110-1L .

What cell culture systems are appropriate for studying MGF 110-1L function?

Primary swine macrophage cultures represent the gold standard for in vitro studies of MGF 110-1L function. These cultures closely mimic the natural host cells for ASFV replication and provide the most physiologically relevant system for functional analyses . When establishing such cultures:

• Cell preparation:

  • Isolate peripheral blood mononuclear cells from swine blood

  • Allow differentiation into macrophages through adherence to culture vessels

  • Culture in appropriate media supplemented with necessary growth factors

• Infection parameters:

  • For multi-step growth curves, use low multiplicity of infection (MOI) (e.g., 0.01) to observe multiple rounds of replication

  • Collect samples at various time points (e.g., 2, 24, 48, 72, and 96 hours post-infection) to assess viral kinetics

• Quantification methods:

  • Hemadsorption assays (HAD50) for quantifying infectious virus

  • Fluorescence microscopy for tracking reporter gene expression

  • qPCR for viral genome quantification

Using these primary cell cultures allows researchers to accurately assess the impact of MGF 110-1L on viral replication, gene expression, and host cell interactions in a relevant cellular context.

What methods can be used to express and purify recombinant MGF 110-1L protein?

Expression and purification of recombinant MGF 110-1L protein can be achieved through the following methodological approach:

• Expression system selection:

  • Bacterial systems (E. coli): Suitable for basic structural studies

  • Eukaryotic systems (insect or mammalian cells): Preferred for functional studies requiring post-translational modifications

  • Cell-free systems: For rapid screening or potentially toxic proteins

• Construct design:

  • Clone the MGF 110-1L coding sequence (e.g., amino acids 1-269 for War-006) into an appropriate expression vector

  • Include purification tags (His, GST, etc.) to facilitate downstream purification

  • Consider codon optimization for the selected expression system

• Purification strategy:

  • Affinity chromatography using tag-specific resins

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography for final polishing and buffer exchange

• Quality control:

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Mass spectrometry for precise molecular characterization

  • Functional assays to verify biological activity

Commercial recombinant ASFV War-006 protein (aa 1-269) is available for research purposes, indicating successful implementation of such expression and purification protocols .

How does deletion of MGF 110-1L affect ASFV replication in vitro?

Comparative growth kinetics studies between wild-type ASFV and MGF 110-1L deletion mutants have yielded significant insights into the protein's role in viral replication. Research using the ASFV-G-ΔMGF110-1L mutant revealed:

• Growth kinetics: The MGF 110-1L deletion mutant displayed similar growth kinetics to the parental ASFV-G strain in primary swine macrophage cultures infected at MOI 0.01 .

• Viral yields: No significant differences in viral titers were observed between ASFV-G-ΔMGF110-1L and ASFV-G at any time point (2, 24, 48, 72, and 96 hours post-infection) .

• Replication competence: The deletion mutant maintained full replication competence, demonstrating that MGF 110-1L is non-essential for viral replication in vitro .

This unexpected finding is particularly intriguing given the universal conservation of MGF 110-1L across all ASFV isolates, suggesting that while the gene may not be essential for basic replication, it might serve important functions in specific contexts not captured in standard cell culture systems .

The paradoxical finding that MGF 110-1L is universally conserved yet apparently dispensable for viral replication and virulence presents an intriguing research question. Several hypotheses can explain this contradiction:

• Functional redundancy: Other MGF110 family genes may compensate for MGF 110-1L loss in experimental settings. The MGF110 family in ASFV-G contains 11 different genes, some of which might provide functional backup .

• Context-dependent importance: MGF 110-1L may be critical in specific hosts or transmission cycles not captured in laboratory experiments. ASFV naturally circulates between soft ticks (Ornithodoros) and wild suids in Africa, environments not replicated in standard research protocols .

• Subtle phenotypes: The deletion may cause subtle fitness defects not detectable in acute infection models but significant in natural transmission cycles or persistent infections .

• Evolutionary constraints: The conservation might reflect historical importance rather than current necessity, or the protein may interact with host factors in ways beneficial but not essential to the virus .

Researchers investigating this paradox should consider experimental designs that:

• Test fitness in multiple cell types and hosts

• Examine competitive fitness between wild-type and deletion mutants

• Investigate transmission efficiency between hosts

• Explore potential roles in natural host-vector cycles

How can recombinant MGF 110-1L be utilized in vaccine development studies?

Recombinant MGF 110-1L offers several applications in ASFV vaccine development research:

• Subunit vaccine candidate: As a conserved viral protein, recombinant MGF 110-1L could be evaluated as a component of subunit vaccines, potentially eliciting cross-protective immune responses against multiple ASFV strains .

• Marker for differentiation: The high conservation of MGF 110-1L makes it a potential target for developing serological tests that can differentiate infected from vaccinated animals (DIVA strategy), particularly if used alongside live-attenuated vaccine candidates lacking this protein .

• Rational attenuation strategy: Although MGF 110-1L deletion alone does not attenuate ASFV-G, it could be combined with other genetic modifications to develop rationally attenuated vaccine candidates with predictable safety profiles .

• Immunogenicity studies: Purified recombinant MGF 110-1L can be used to assess specific immune responses in vaccinated or infected animals, helping characterize potential protective mechanisms .

• Adjuvant formulation testing: The protein can serve as a model antigen for testing various adjuvant formulations and delivery systems to enhance ASFV-specific immune responses .

While MGF 110-1L deletion alone is insufficient for attenuation, its universal conservation makes it valuable for understanding fundamental aspects of ASFV immunity and developing cross-protective vaccination strategies .

What role can MGF 110-1L play in diagnostic development for ASFV?

The highly conserved nature of MGF 110-1L presents several opportunities for diagnostic applications:

• Serological assays: Recombinant MGF 110-1L can be used as a capture antigen in ELISA or other serological assays to detect ASFV-specific antibodies . The protein's conservation across all ASFV isolates makes it an ideal candidate for developing broadly reactive diagnostic tests.

• PCR-based detection: The conserved sequences within the MGF 110-1L gene provide potential targets for designing universal PCR primers and probes for ASFV detection across diverse isolates .

• Protein-based detection: Antibodies raised against recombinant MGF 110-1L could be employed in antigen-capture assays for direct virus detection in clinical samples .

• Multiplex assay panels: MGF 110-1L-based assays could be incorporated into multiplex diagnostic panels alongside tests targeting other conserved ASFV proteins to improve diagnostic sensitivity and specificity .

• Field-applicable diagnostics: The conservation of MGF 110-1L makes it suitable for inclusion in point-of-care or field-deployable diagnostic platforms, which are especially valuable in endemic regions with limited laboratory infrastructure .

The universal presence of MGF 110-1L across ASFV isolates provides a significant advantage for diagnostic applications requiring broad detection capabilities across diverse viral strains .

How can MGF 110-1L be used to study ASFV-host interactions?

MGF 110-1L provides several experimental opportunities for investigating ASFV-host interactions:

• Protein interaction studies: Recombinant MGF 110-1L can be used in pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens to identify host cellular factors that interact with this viral protein .

• Localization studies: Using tagged versions of MGF 110-1L in infected or transfected cells allows researchers to determine its subcellular localization and potential co-localization with host factors during infection .

• Temporal expression analysis: The early expression pattern of MGF 110-1L suggests potential roles in modulating early host responses. Time-course studies combining transcriptomics and proteomics can reveal correlations between MGF 110-1L expression and changes in host gene expression .

• Comparative studies: The availability of MGF 110-1L deletion mutants permits direct comparison of host responses to wild-type and mutant viruses, potentially revealing specific host pathways affected by this protein .

• Cross-species comparisons: Given that ASFV naturally infects both soft ticks and various suids with different disease outcomes, studying MGF 110-1L's interactions with host factors across species may provide insights into its potential role in host adaptation .

While current evidence indicates MGF 110-1L is non-essential for viral replication and virulence in domestic pigs, its universal conservation suggests it may play important roles in specific host contexts or transmission cycles that remain to be elucidated .

What are the primary knowledge gaps in understanding MGF 110-1L function?

Despite significant progress in characterizing MGF 110-1L, several critical knowledge gaps remain:

• Molecular function: The precise molecular function of MGF 110-1L remains undefined. No enzymatic activities, binding partners, or specific cellular pathways modulated by this protein have been conclusively identified .

• Structural information: Detailed three-dimensional structural data for MGF 110-1L is lacking, limiting structure-based functional predictions and rational design of inhibitors or vaccines .

• Expression in natural hosts: While MGF 110-1L expression has been characterized in cell culture, its expression patterns and levels in natural hosts (including wild suids and tick vectors) remain largely unexplored .

• Evolutionary history: Despite its conservation, the evolutionary history and selective pressures acting on MGF 110-1L across different ASFV lineages are not fully understood .

• Role in tick hosts: ASFV naturally cycles between ticks and wild suids in Africa, but the function of MGF 110-1L in tick infection or transmission has not been investigated .

Addressing these knowledge gaps will require multidisciplinary approaches combining structural biology, comparative genomics, experimental infections in natural hosts, and advanced molecular techniques to elucidate the true biological significance of this enigmatic protein .

What methodological innovations could advance MGF 110-1L research?

Several methodological innovations could significantly accelerate research on MGF 110-1L:

• CRISPR-Cas9 genome editing: Implementing CRISPR-based approaches for more precise and efficient genetic manipulation of ASFV could facilitate functional studies through targeted mutations rather than complete gene deletions .

• Single-cell transcriptomics/proteomics: Applying single-cell analyses to ASFV-infected cultures could reveal cell-to-cell variation in MGF 110-1L expression and effects, potentially identifying specialized roles in subpopulations of infected cells .

• Organoid models: Developing swine macrophage organoids or more complex tissue models could provide more physiologically relevant systems for studying MGF 110-1L function beyond conventional cell cultures .

• Cryo-electron microscopy: Applying cryo-EM techniques to determine the structure of MGF 110-1L alone and in complex with potential binding partners would provide crucial insights into its functional mechanisms .

• Comparative functional genomics: Systematic comparison of MGF 110-1L function across multiple ASFV strains with varying virulence could identify strain-specific variations in protein function relevant to pathogenesis .

• Tick infection models: Developing standardized methods to study ASFV infection in its arthropod host could reveal potential roles for MGF 110-1L in the tick phase of the viral life cycle .

These methodological advances would help resolve the paradox between MGF 110-1L's universal conservation and its apparent dispensability in experimental systems .

What promising research directions might advance our understanding of MGF 110-1L?

Several promising research directions could significantly enhance our understanding of MGF 110-1L:

• Comparative virulence studies: Constructing and testing MGF 110-1L deletion mutants in naturally attenuated ASFV strains might reveal context-dependent functions not apparent in highly virulent backgrounds like ASFV-G .

• Long-term persistence models: Investigating the role of MGF 110-1L in establishing or maintaining persistent infections in recovered animals could reveal functions relevant to viral persistence rather than acute virulence .

• Host range studies: Examining the impact of MGF 110-1L deletion on ASFV replication in cells derived from different host species (domestic pigs, warthogs, bushpigs) could identify host-specific functions .

• Tick-pig transmission cycle: Studying the role of MGF 110-1L in the complete natural transmission cycle, including tick vectors, might reveal important functions in interspecies transmission .

• Combination gene deletion studies: Creating mutants lacking MGF 110-1L along with related MGF genes could overcome potential functional redundancy and reveal phenotypes not apparent with single deletions .

• Competitive fitness assays: Direct competition experiments between wild-type virus and MGF 110-1L deletion mutants in mixed infections could reveal subtle fitness effects masked in standard single-infection models .