Recombinant African swine fever virus Protein MGF 110-1L (Pret-008)

What is the evolutionary significance of MGF 110-1L in African swine fever virus?

MGF 110-1L holds a unique evolutionary position as the only member of the MGF 110 family that is present in all sequenced ASFV isolates, suggesting it may have a conserved role in viral biology . The protein shows high sequence conservation, particularly within genotype II viruses where there is 100% amino acid identity for the first 196 amino acids . This conservation pattern across diverse ASFV isolates indicates potential functional importance, despite experimental evidence suggesting it is non-essential for viral replication or virulence. Evolutionary analyses reveal that while most non-conserved residues tend to be conserved within specific genotypes, approximately 84% of all residues in the ASFV-G protein sequence are conserved across all isolates . This pattern of conservation might reflect selective pressure maintaining this gene throughout ASFV evolution.

What is known about the structural characteristics of the MGF 110-1L protein?

The MGF 110-1L protein varies in length across different ASFV isolates, ranging from 196 to 271 amino acids . This size variation primarily occurs due to extensions at the C-terminus of longer protein variants. The ASFV Georgia 2007 (ASFV-G) strain produces a 214 amino acid MGF 110-1L protein, while other genotype II isolates may encode versions with different lengths, such as the Kashino isolate which produces a 269 amino acid protein . Structurally, the protein appears to have conserved domains across the various isolates, though detailed crystallographic data is not currently available in the provided research. Functional domains and motifs that might explain its biological role have not been fully characterized, representing a significant knowledge gap in our understanding of this ubiquitous viral protein.

What are the recommended methods for generating MGF 110-1L deletion mutants?

Based on published research, successful generation of MGF 110-1L deletion mutants can be accomplished through homologous recombination between the parental ASFV genome and a recombination transfer vector . The methodology involves:

• Construction of a recombination transfer vector containing:

  • Left arm homologous to the genomic region upstream of MGF 110-1L (typically 1000bp)

  • Right arm homologous to the genomic region downstream of MGF 110-1L (typically 1000bp)

  • A reporter gene cassette (e.g., mCherry fluorescent protein gene under the control of the ASFV p72 late gene promoter)

• Infection of macrophage cell cultures with the parental ASFV strain followed by transfection with the recombination transfer vector

• Purification of the deletion mutant through successive rounds of limiting dilution, selecting for cells showing the reporter gene expression

• Confirmation of the deletion and genome integrity through next-generation sequencing

In the published example, researchers deleted a 645-nucleotide region between positions 7004-7648 of the ASFV genome, effectively removing the complete MGF 110-1L ORF and replacing it with a p72mCherry cassette . This approach ensures complete deletion of the target gene while providing a visual marker for the selection of recombinant viruses.

What experimental controls should be included when evaluating MGF 110-1L function?

When investigating MGF 110-1L function, several critical controls should be incorporated:

• Parental virus control: Always include the unmodified parental virus (e.g., ASFV-G) as a positive control in all experiments to establish baseline viral characteristics

• Growth kinetics comparison: Conduct multi-step growth curves comparing the deletion mutant with the parental virus in primary swine macrophage cultures at standardized MOI (e.g., 0.01) with sampling at appropriate timepoints (2, 24, 48, 72, and 96 hours post-infection)

• Full genome sequencing: Confirm that no unintended mutations were introduced during the recombination process by sequencing the entire genome of the deletion mutant and comparing it to the parental strain

• Contamination assessment: Verify the absence of any residual parental virus in the deletion mutant stock through NGS analysis

• In vivo controls: When conducting animal experiments, include groups inoculated with the parental virus via the same route and dose to allow direct comparison of clinical outcomes, viremia levels, and pathological findings

These controls ensure that any observed differences in viral behavior can be attributed specifically to the deletion of MGF 110-1L rather than to unintended genetic alterations or experimental variables.

How does deletion of MGF 110-1L affect ASFV virulence in swine models?

Experimental evidence indicates that deletion of MGF 110-1L does not significantly alter ASFV virulence in domestic swine . When pigs were intramuscularly infected with the ASFV-G-ΔMGF110-1L deletion mutant, they developed a clinical disease pattern indistinguishable from that caused by the parental ASFV-G strain . Key findings include:

• Clinical signs: Animals infected with either virus developed similar clinical manifestations, including fever, anorexia, depression, purple skin discoloration, and staggering gait

• Viremia levels: Pigs infected with ASFV-G-ΔMGF110-1L showed viremia values of 10^6-10^6.8 HAD50/mL by day 4 post-infection, reaching similar titers to those of animals infected with the parental ASFV-G by day 7

• Disease progression: The deletion mutant maintained the high virulence characteristic of the parental strain, with all animals requiring euthanasia by day 7-8 post-infection

This surprising finding suggests that despite MGF 110-1L being the only MGF 110 family gene conserved across all ASFV isolates, it is not a critical determinant of viral virulence in domestic swine . The maintenance of virulence despite the deletion suggests functional redundancy within the viral genome, possibly from other MGF 110 genes or other ASFV proteins that may compensate for its absence.

What are the comparative clinical and pathological findings between parental and MGF 110-1L deletion mutant ASFV strains?

Comparative studies between parental ASFV strains and MGF 110-1L deletion mutants have revealed striking similarities in clinical presentation and pathology:

Table 1: Comparison of Clinical and Pathological Findings

These findings consistently demonstrate that deletion of MGF 110-1L does not substantially alter the pathogenesis of ASFV in domestic pigs . The preservation of the full virulent phenotype despite the absence of this highly conserved gene raises important questions about its actual function in the viral life cycle and disease pathogenesis.

How does MGF 110-1L deletion affect viral replication kinetics in different cell types?

Studies examining the replication of MGF 110-1L deletion mutants in primary swine macrophage cultures have shown no significant differences compared to parental virus strains . In multi-step growth curve experiments (MOI of 0.01), ASFV-G-ΔMGF110-1L displayed similar growth kinetics to the parental ASFV-G strain across all time points examined (2, 24, 48, 72, and 96 hours post-infection) .

• The replication kinetics have primarily been studied in primary swine macrophages, leaving questions about potential cell-type specific effects

• Potential subtle differences in replication efficiency that might only become apparent under specific stress conditions or in different macrophage activation states have not been thoroughly investigated

• The impact of MGF 110-1L deletion on viral replication in tick cells or other potential reservoir hosts has not been reported

These gaps present opportunities for future research to determine if MGF 110-1L might play more subtle roles in viral replication under specific conditions or in alternative host systems.

What is known about the transcriptional regulation and expression timing of MGF 110-1L?

MGF 110-1L is transcribed as an early viral gene during the ASFV replication cycle . Analysis of transcriptional activity through microarray data has revealed:

• MGF 110-1L transcripts are detectable at all examined time points post-infection

• Expression follows a pattern similar to other early viral genes, like p30 (CP204L)

• The expression pattern shows an initial gradual decrease from 3 to 9 hours post-infection, followed by an increase from 12 to 18 hours post-infection

What cellular pathways or host interactions might MGF 110-1L potentially influence?

While the specific function of MGF 110-1L remains uncharacterized, insights can be drawn from research on other MGF 110 family proteins:

• MGF 110-4L and -6L localize to pre-Golgi compartments and may be involved in endoplasmic reticulum (ER) rearrangements that impair cytokine production or antigen presentation

• MGF 110-7L activates the PERK/PKR-IF2a pathway, influencing host gene translation and inhibiting stress granule formation

• MGF 110-9L deletion from a highly virulent strain results in partial attenuation, suggesting some role in virulence

The protein may interact with host factors that are important in natural host-virus relationships but are not critical in the experimental pig infection model. Alternatively, it might provide advantages in circumstances not replicated in laboratory settings, such as transmission efficiency, persistence in the environment, or replication in arthropod vectors.

What approaches can be used to identify binding partners and functional roles of MGF 110-1L?

To elucidate the function of MGF 110-1L, researchers should consider employing multiple complementary approaches:

• Proteomic identification of binding partners:

  • Immunoprecipitation followed by mass spectrometry

  • Proximity labeling methods (BioID or TurboID) to identify proteins in close proximity

  • Yeast two-hybrid screening against swine and tick cell protein libraries

• Subcellular localization studies:

  • Fluorescent protein tagging to determine localization during infection

  • Immunofluorescence with specific antibodies against MGF 110-1L

  • Fractionation studies to identify what cellular compartments contain the protein

• Transcriptomic/proteomic comparisons:

  • RNA-seq analysis comparing host responses to wild-type and ΔMGF110-1L viruses

  • Temporal proteomics to identify cellular pathways differentially regulated

• Functional rescue experiments:

  • Complementation studies with MGF 110-1L from different ASFV isolates

  • Domain mapping through truncation or point mutation analyses

  • Heterologous expression to determine if specific cellular phenotypes can be induced

• Alternative infection models:

  • Examination of virus behavior in natural reservoir hosts (warthogs)

  • Assessment of replication and transmission in tick vectors

  • Long-term persistence studies that might reveal subtle phenotypes

These approaches would provide a comprehensive understanding of MGF 110-1L's role in the ASFV life cycle and potentially explain the evolutionary conservation of this apparently non-essential gene.

What is the potential utility of MGF 110-1L deletion or modification in ASFV vaccine development?

While current evidence suggests that deletion of MGF 110-1L alone does not attenuate ASFV virulence , its study contributes to the broader understanding of ASFV biology that may inform vaccine development strategies:

• As part of combination deletions: While MGF 110-1L deletion alone does not attenuate virulence, it could potentially be combined with other genetic modifications to create rationally designed attenuated vaccine candidates

• Knowledge contribution: Understanding conserved viral proteins like MGF 110-1L helps build the fundamental knowledge base necessary for rational vaccine design

• Vector development: The non-essential nature of MGF 110-1L suggests its locus could potentially serve as an insertion site for foreign antigens in viral vector-based vaccines

• Diagnostic differentiation: MGF 110-1L deletion mutants could potentially be utilized in DIVA (Differentiating Infected from Vaccinated Animals) strategies, where diagnostic tests could distinguish between vaccination and natural infection

Although the MGF 110-11L gene (distinct from MGF 110-1L) has been explored as a deletion target to improve usability of attenuated ASFV strains as vaccines , MGF 110-1L itself appears unlikely to be a primary target for single-gene deletion approaches to vaccine development based on current evidence showing its deletion does not reduce virulence .

How might the conservation of MGF 110-1L across ASFV isolates impact cross-protection strategies?

The high conservation of MGF 110-1L across all ASFV isolates has several implications for cross-protection strategies:

• Potential as a universal ASFV antigen: The conservation of MGF 110-1L (84% residue conservation across all isolates ) suggests it could potentially serve as a broadly recognized antigen across different ASFV genotypes

• Epitope mapping opportunities: Identifying conserved epitopes within MGF 110-1L could inform the development of subunit or epitope-based vaccines with broader protection

• Evolutionary constraints: The conservation pattern indicates evolutionary constraints on MGF 110-1L sequence, suggesting that immune targeting of this protein might be less likely to drive escape mutations

• Diagnostic applications: The conserved nature makes MGF 110-1L a potential target for broadly reactive diagnostic assays that could detect diverse ASFV strains

What explains the evolutionary conservation of MGF 110-1L despite its apparent dispensability for virulence?

The paradoxical conservation of MGF 110-1L across all ASFV isolates despite experimental evidence showing it is dispensable for replication and virulence presents an intriguing research puzzle . Several hypotheses could explain this phenomenon:

• Function in natural ecological contexts: MGF 110-1L might be critical for aspects of the virus life cycle not captured in experimental settings, such as:

  • Transmission between hosts

  • Survival in arthropod vectors

  • Persistence in natural reservoir hosts

  • Environmental stability

• Subtle fitness advantages: The protein may confer subtle advantages that become significant over evolutionary time but are not apparent in short-term laboratory experiments

• Functional redundancy with conditional requirements: Other viral proteins may compensate for MGF 110-1L function under experimental conditions, but this redundancy might fail under specific natural circumstances

• Host-specific functions: MGF 110-1L might interact with specific host factors present in the natural hosts (warthogs, bushpigs) or vectors (Ornithodoros ticks) but absent or different in domestic pigs

• Genomic stability role: The gene or its locus might play a role in maintaining genomic stability or proper gene expression of neighboring genes

Testing these hypotheses would require broader ecological and evolutionary approaches beyond traditional virulence studies, potentially including field studies in endemic regions with natural host species.

How might MGF 110-1L interact with other multigene family proteins in the context of ASFV infection?

The potential interactions between MGF 110-1L and other multigene family proteins represent an important area for future research:

• Functional redundancy: The dispensability of MGF 110-1L for virulence despite its conservation suggests possible functional redundancy with other MGF proteins . Research could explore whether deletion of MGF 110-1L along with related family members produces more pronounced phenotypes.

• Protein complexes: MGF 110-1L might function within protein complexes containing other viral proteins, including other MGF family members. Proteomic approaches could identify such interactions.

• Regulatory relationships: The expression of MGF 110-1L might influence the expression or function of other viral genes, creating interdependencies that are not apparent when examining single-gene deletions.

• Evolutionary co-adaptation: Sequence analysis across multiple ASFV isolates could reveal patterns of co-evolution between MGF 110-1L and other viral proteins, potentially identifying functional relationships.

• Comparative analyses across MGF families: Systematic comparison of phenotypes resulting from deletions of different MGF family genes could reveal functional patterns and relationships between these gene families.

Understanding these potential interactions would provide a more complete picture of ASFV biology and might reveal emergent properties not apparent from studying individual genes in isolation.