Recombinant African swine fever virus Protein MGF 110-1L (BA71V-008)

What is MGF 110-1L and how is it characterized in the ASFV genome?

MGF 110-1L is a member of the multi-gene family 110 (MGF 110) encoded by the African swine fever virus. ASFV contains four multi-gene family groups that have been implicated in regulating the immune response and host specificity . The MGF 110-1L gene is part of a larger genomic structure where ASFV encodes more than 160 genes within its large double-stranded DNA genome (170-193 kb in length) . This protein is identified in various ASFV isolates, including the BA71V strain, which is a cell culture-adapted attenuated variant of the virulent BA71 isolate .

Methodologically, researchers characterize this gene through genome sequencing and comparative genomics approaches. Profile hidden Markov model (HMM) domain analysis has emerged as a valuable alignment-free method to document viral diversity and identify functional protein domains in different ASFV strains .

What expression systems are used for producing recombinant MGF 110-1L protein?

Recombinant MGF 110-1L is typically expressed in heterologous systems, with E. coli being a common expression platform . The protocol generally involves:

• Cloning the MGF 110-1L gene sequence into an appropriate expression vector

• Transformation into competent E. coli cells

• Induction of protein expression

• Cell lysis and protein purification using affinity chromatography

For the specific recombinant ASFV MGF 110-1L Protein (aa 1-269) from isolate Tick/South Africa/Pretoriuskop Pr4/1996, the protein has been successfully expressed in E. coli systems with a Uniprot ID of P0C9G4 . Researchers must ensure proper folding and solubility of the expressed protein, as these factors significantly affect downstream applications.

What are the known functional characteristics of MGF 110-1L?

Research indicates that MGF 110-1L is non-essential for viral replication. Deletion mutants lacking this gene (ASFV-G-ΔMGF110-1L) demonstrate similar replication kinetics in primary swine macrophage cell cultures when compared to the parental virulent isolate Georgia2007 (ASFV-G) . Experimental infections of domestic pigs with ASFV-G-ΔMGF110-1L produced clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the MGF 110-1L gene does not significantly impact viral virulence .

This finding contrasts with other MGF genes, particularly those in the MGF 360 and MGF 505 families, which have been associated with changes in virulence when deleted . Domain analysis techniques have been valuable in identifying these functional differences between virulent and attenuated strains.

What gene editing approaches are most effective for studying MGF 110-1L function?

For studying MGF 110-1L function, recombination-based gene deletion has proven effective, as demonstrated in the development of the ASFV-G-ΔMGF110-1L mutant . The methodology typically involves:

• Construction of a recombination transfer vector containing flanking regions of the MGF 110-1L gene

• Homologous recombination in infected cells to replace the target gene

• Selection and purification of recombinant viruses

• Verification of deletion through PCR and sequencing

• In vitro characterization in primary swine macrophage cultures

• In vivo evaluation in susceptible animals

This approach allows researchers to directly assess the contribution of MGF 110-1L to viral replication, host range, and pathogenesis through comparative studies with the parental virus.

How does MGF 110-1L sequence variation compare across ASFV isolates?

ASFV isolates show considerable genetic variation, with differences observed in the number of copies of multigene families, including MGF 110. Genomic analysis techniques can reveal:

Selection pressure analysis indicates that some ASFV genes are under positive diversifying selection, representing elevated amino acid diversity within or across viral populations . This evolutionary pressure can potentially influence the functional properties of MGF proteins, including MGF 110-1L.

What is the impact of MGF 110-1L on host immune responses?

While MGF 110-1L deletion does not significantly affect virulence in experimental infections , other multigene family members, particularly from MGF 360 and MGF 505 families, have been implicated in modulating host immune responses. Research methodologies to investigate immune modulation by MGF proteins typically include:

• Transcriptomic analysis of infected cells with wild-type versus deletion mutants

• Measurement of type I interferon responses

• Assessment of inflammatory cytokine production

• Evaluation of immune cell recruitment and activation

• Protein-protein interaction studies to identify host targets

The specific role of MGF 110-1L in immune modulation requires further investigation to determine if it contributes subtly to immune evasion strategies employed by ASFV.

How can structural analysis of MGF 110-1L inform vaccine development efforts?

The methodological approach includes:

• Secondary structure prediction using tools such as PSIpred or PROMALS3D

• Identification of conserved domains across isolates

• Mapping of non-synonymous mutations to protein domain architecture

• Assessment of immunogenic epitopes

• Evaluation of protein accessibility in the viral particle

Research shows that some ASFV proteins induce immune responses in swine following ASFV challenge, and structural analysis can help identify potential antigenic regions for vaccine targeting .

What experimental systems best model MGF 110-1L function in the context of host-pathogen interactions?

The optimal experimental systems for studying MGF 110-1L function include:

• Primary swine macrophage cultures: These represent the natural host cell target and provide the most relevant cellular context for studying ASFV replication and host interactions .

• Experimental infection of domestic pigs: This approach allows for assessment of virulence, clinical progression, and immune responses in the natural host species .

• Comparative genomic analysis: Using profile HMM domain analysis to identify patterns of genomic variation between virulent and attenuated strains provides insights into functional domains .

• Recombinant protein expression systems: For biochemical and structural studies, purified recombinant proteins expressed in heterologous systems like E. coli allow for targeted functional analyses .

Each system offers distinct advantages, and a comprehensive understanding of MGF 110-1L function likely requires integration of data from multiple experimental approaches.

What are the challenges in expressing and purifying functionally active recombinant MGF 110-1L?

Researchers face several challenges when working with recombinant MGF 110-1L:

• Protein solubility: Viral proteins often form inclusion bodies in bacterial expression systems, requiring optimization of expression conditions or refolding protocols.

• Post-translational modifications: Bacterial systems lack the machinery for eukaryotic post-translational modifications that might be essential for protein function.

• Protein structure preservation: Maintaining the native conformation during purification is critical for functional studies.

• Protein stability: Long-term storage without activity loss requires optimization of buffer conditions.

Using tagged protein versions (His-tag, GST-tag) facilitates purification but may interfere with protein function, necessitating tag removal in some applications .

How can genetic manipulation of MGF 110-1L contribute to understanding ASFV pathogenesis?

Although deletion of MGF 110-1L alone does not significantly alter viral virulence , genetic manipulation of this gene in combination with other viral factors may provide insights into ASFV pathogenesis. Research approaches include:

• Multiple gene deletion studies to identify synergistic effects

• Point mutations to assess specific amino acid contributions to protein function

• Domain swapping between virulent and attenuated strains

• Insertion of reporter genes to track protein localization and interactions

• Construction of chimeric viruses to map virulence determinants

These approaches can help unravel the complex interactions between viral proteins and host factors that determine ASFV pathogenicity.

What emerging technologies could advance MGF 110-1L research?

Emerging technologies with potential to advance MGF 110-1L research include:

• CRISPR-Cas9 genome editing: For more precise manipulation of the ASFV genome

• Cryo-electron microscopy: To determine high-resolution protein structures

• Single-cell transcriptomics: To identify cell-specific responses to viral proteins

• Protein-protein interaction mapping: To identify host targets of MGF 110-1L

• Systems biology approaches: To integrate diverse datasets for comprehensive understanding of protein function

Application of these technologies could overcome current limitations in understanding the specific roles of MGF 110-1L in ASFV biology and host interactions.

How might MGF 110-1L research inform control strategies for African swine fever?

While MGF 110-1L deletion does not attenuate ASFV , research on this protein contributes to the broader understanding of ASFV biology necessary for developing effective control strategies. Key contributions include:

• Expanding knowledge of ASFV genome organization and evolution

• Identifying viral factors that may contribute to host range or tissue tropism

• Understanding the genetic basis for differences between field isolates

• Supporting rational approaches to attenuated vaccine development

The comprehensive characterization of ASFV proteins, including MGF 110-1L, is essential for developing the next generation of diagnostic tools, antiviral therapies, and vaccines against this economically devastating disease .