Recombinant African swine fever virus Protein MGF 110-1L (Mal-005)

What is MGF 110-1L and what is its role in the ASFV genome?

MGF 110-1L is one of the multi-gene family (MGF) proteins encoded by 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 encodes a protein of approximately 214 amino acid residues and is highly conserved among ASFV isolates . It belongs to the MGF110 family, which is present in all sequenced ASFV isolates. The protein is expressed early during viral infection, suggesting a potential role in the early stages of the viral life cycle .

What is known about the structure and sequence of the MGF 110-1L protein?

The MGF 110-1L protein is approximately 269 amino acids in length. According to research, the full amino acid sequence includes: "MLGLQIFTLLSIPTLLYTYELEPLERTSTLPEKELGYWCTYANHCRFCWDCQDGICRNKAFKNHSPILENDYIANCSVYRSNNFCIYYITSIKPHKMYRTECPQYMSHEWHEAVIRKWQKLLTYGFYLVGCVLVANYVRKRSLQTIMYLMVLLVIFFLLSQLMLYRELEDKKHKIGSIPPERELEHWCTHGKYCNFCWDCQNGICKNKVFKNHPPIGENDFIRYDCWTTHLLNKCNYEKIYKHFDTHIMECSQPTHFKWYDNLMKKQDM" . The protein contains multiple cysteine residues that may be involved in structural stability through disulfide bonding, though detailed structural studies are still limited.

How is recombinant MGF 110-1L protein produced for research purposes?

For research applications, recombinant MGF 110-1L protein is typically produced using bacterial expression systems. The recombinant protein is often expressed in E. coli with an N-terminal His-tag to facilitate purification . The production process involves:

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

• Transforming E. coli with the construct

• Inducing protein expression under optimized conditions

• Purifying the protein using affinity chromatography (taking advantage of the His-tag)

• Processing the purified protein into a stable form, often as a lyophilized powder in a buffer containing trehalose to maintain stability

What experimental evidence exists regarding the essentiality of MGF 110-1L for ASFV replication and virulence?

Extensive in vitro and in vivo studies have investigated the role of MGF 110-1L in ASFV biology. A deletion mutant virus (ASFV-G-ΔMGF110-1L) was constructed by removing a 645-nucleotide region (positions 7004-7648) from the ASFV Georgia 2007 isolate (ASFV-G) genome and replacing it with a p72mCherry reporter gene cassette .

The deletion mutant demonstrated that MGF 110-1L is non-essential for virus replication in vitro. When compared to the parental ASFV-G in multi-step growth curves in swine macrophage cultures, ASFV-G-ΔMGF110-1L displayed similar replication kinetics . This was somewhat surprising considering the gene's conservation across all ASFV genomes, suggesting potential functional redundancy with other MGF110 family genes.

In vivo experimental infections of domestic pigs with ASFV-G-ΔMGF110-1L produced a clinical disease pattern indistinguishable from the parental ASFV-G, with animals developing similar clinical signs, pathology, and viremia levels (10^6-10^7.85 HAD50/mL). This evidence confirms that deletion of the MGF110-1L gene does not affect viral virulence in swine .

How can researchers verify the successful deletion of MGF 110-1L in recombinant virus strains?

Researchers can verify MGF 110-1L deletion using several complementary approaches:

• Next-Generation Sequencing (NGS): Full genome sequencing using platforms like Illumina NextSeq500 with analysis via software such as CLC Genomics Workbench to confirm the deletion and insertion of reporter cassettes. This approach can verify both the targeted deletion and ensure no unintended mutations were introduced during the recombination process .

• Fluorescence microscopy: If a fluorescent reporter (like mCherry) is used to replace the deleted gene, direct visualization of infected cells can confirm the presence of the recombinant virus .

• PCR verification: Primers flanking the deletion site can be used to amplify the region, with the product size indicating whether the gene is present or deleted.

• Real-time PCR: Quantitative PCR targeting the MGF 110-1L gene and comparing Ct values between samples can demonstrate the absence of the gene in deletion mutants .

What methods are most effective for studying MGF 110-1L protein function in vitro?

Several methodological approaches have proven effective for investigating MGF 110-1L function:

• Gene deletion and phenotypic analysis: Creating recombinant viruses lacking MGF 110-1L through homologous recombination, followed by comparative analysis of replication kinetics in primary swine macrophage cultures .

• Growth kinetics analysis: Multi-step growth curves with sampling at various time points (2, 24, 48, 72, and 96 hours post-infection) to assess viral replication efficiency in the presence or absence of MGF 110-1L .

• Protein-protein interaction studies: Using recombinant His-tagged MGF 110-1L protein to identify potential cellular binding partners through pull-down assays.

• Immunolocalization: Using antibodies against MGF 110-1L to determine its subcellular localization during different stages of viral infection.

• Functional complementation: Reintroducing the MGF 110-1L gene into deletion mutants to confirm phenotype restoration, which can validate that observed effects are specifically due to the absence of this gene.

How should researchers design experiments to assess potential immune evasion functions of MGF 110-1L?

While current research suggests MGF 110-1L is not essential for virulence, it may still play a role in immune evasion that requires specific experimental designs to detect:

• Comparative transcriptomics: Compare host gene expression profiles in macrophages infected with wild-type virus versus MGF 110-1L deletion mutants to identify differentially regulated immune pathways.

• Cytokine profiling: Measure production of key inflammatory cytokines (IFN-α, IFN-β, IL-1β, TNF-α) in response to infection with wild-type versus deletion mutant viruses.

• Pattern recognition receptor activation: Assess the activation of key innate immune sensors (RIG-I, MDA5, cGAS-STING) in the presence or absence of MGF 110-1L.

• Host protein degradation assays: Determine if MGF 110-1L targets specific host immune factors for degradation by comparing their stability in cells expressing or lacking the viral protein.

• In vivo immune response kinetics: Monitor temporal development of both innate and adaptive immune responses in animals infected with wild-type versus MGF 110-1L deletion mutants.

What is the recommended protocol for creating MGF 110-1L knockout ASFV for functional studies?

Based on successful previous research, the following protocol is recommended:

• Design and construct a recombination transfer vector containing:

  • Left arm homology region (approximately 1000bp upstream of MGF 110-1L)

  • Right arm homology region (approximately 1000bp downstream of MGF 110-1L)

  • Reporter gene cassette (e.g., p72mCherry) for selection

• Perform homologous recombination:

  • Infect macrophage cultures with parental ASFV

  • Transfect infected cells with the recombination transfer vector

  • Allow recombination to occur during viral replication

• Purify recombinant viruses:

  • Use limiting dilution purification

  • Select wells showing mCherry fluorescence at the highest dilution

  • Repeat purification until homogeneous recombinant virus is obtained

• Verify deletion:

  • Extract viral DNA from purified virus stocks

  • Perform full-genome NGS sequencing

  • Confirm deletion of target gene and absence of unwanted mutations

  • Verify absence of parental virus contamination

• Amplify verified recombinant virus:

  • Grow in primary swine macrophage cultures

  • Create standardized virus stocks for experiments

What factors should be considered when using recombinant MGF 110-1L protein in binding studies?

When designing binding studies with recombinant MGF 110-1L protein, researchers should consider:

• Protein conformation: The E. coli-expressed recombinant protein may lack post-translational modifications present in the native viral context. Consider using mammalian expression systems for studies where these modifications might be critical.

• Storage and handling: The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol added for long-term storage at -20°C/-80°C. Repeated freeze-thaw cycles should be avoided .

• Buffer conditions: The protein is typically stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 . Consider the compatibility of this buffer with your binding assay and adjust if necessary.

• Tag interference: The N-terminal His-tag may interfere with certain interactions. Control experiments with tag-cleaved protein or alternatively tagged constructs may be necessary.

• Validation controls: Include positive and negative controls to verify the specificity of any observed interactions.

How should researchers interpret the absence of virulence attenuation in MGF 110-1L deletion mutants?

The finding that MGF 110-1L deletion does not affect ASFV virulence requires careful interpretation:

• Functional redundancy: The MGF 110-1L function may be compensated by other MGF110 family genes or other viral factors. This redundancy is common in large DNA viruses and may explain why deletion of a conserved gene shows no apparent phenotype .

• Context-dependent function: The role of MGF 110-1L may only become apparent under specific conditions not replicated in standard laboratory infections, such as in different host genetic backgrounds or co-infection scenarios.

• Subtle phenotypes: The deletion may cause subtle effects that aren't detected in standard virulence assays. More sensitive readouts or longer-term studies might be necessary.

• Evolutionary pressure: Despite being dispensable in laboratory settings, the consistent conservation of MGF 110-1L across ASFV isolates suggests selective pressure for its maintenance, pointing to an important but as-yet-unidentified function .

What comparative virological data exists between different MGF deletion mutants in ASFV?

Various MGF deletion mutants have been studied in ASFV, providing comparative insights:

These comparative data suggest that while single MGF110 gene deletions may not affect viral properties, multiple deletions within the same family or across different MGF families can have significant effects on viral fitness and virulence.

How can researchers distinguish between direct and indirect effects when studying MGF 110-1L function?

To differentiate direct from indirect effects of MGF 110-1L:

• Complementation studies: Reintroduce the wild-type MGF 110-1L gene into deletion mutants to confirm direct causality if the phenotype is restored.

• Time-course experiments: Determine temporal relationships between MGF 110-1L expression and observed phenotypes. Direct effects should closely follow protein expression.

• Protein-protein interaction verification: For any potential interacting partners identified, verify direct binding using multiple methods (pull-down, co-immunoprecipitation, FRET/BRET).

• Structure-function analysis: Create point mutants or domain deletions to map specific functions to regions of the protein, allowing more precise attribution of effects.

• Heterologous expression systems: Express MGF 110-1L in isolation in different cell types to determine if it can reproduce certain phenotypes independent of other viral factors.

What are the common challenges in generating MGF 110-1L deletion mutants and how can they be overcome?

Researchers may encounter several technical challenges when creating MGF 110-1L deletion mutants:

• Low recombination efficiency:

  • Solution: Optimize transfection conditions for macrophage cultures and consider using electroporation rather than chemical transfection methods.

  • Solution: Ensure high-quality, purified plasmid DNA for the recombination vector.

• Difficulty in purifying recombinant viruses:

  • Solution: Use fluorescent markers like mCherry under control of viral promoters to facilitate identification and purification of recombinant viruses .

  • Solution: Employ multiple rounds of limiting dilution purification, selecting the highest dilution showing reporter gene expression .

• Genome instability:

  • Solution: Verify the stability of the deletion through multiple passages using PCR and sequencing.

  • Solution: Perform full-genome sequencing to ensure no unintended mutations or rearrangements occurred .

• Contamination with parental virus:

  • Solution: Use NGS to verify the purity of the final virus stock and absence of parental virus sequences .

  • Solution: Develop PCR assays specifically targeting the deleted region to screen for contamination.

What quality control measures should be implemented when working with recombinant MGF 110-1L protein?

To ensure experimental reproducibility with recombinant MGF 110-1L protein:

• Purity assessment: Verify protein purity using SDS-PAGE. The commercial preparation typically exceeds 90% purity .

• Functional validation: Develop and implement functional assays specific to the expected activity of the protein.

• Stability monitoring:

  • Avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for no more than one week

  • Store long-term aliquots at -20°C/-80°C with 5-50% glycerol

• Batch consistency: When using commercially sourced protein, request certificate of analysis and compare lot-to-lot variation in critical experiments.

• Endotoxin testing: For cellular assays, verify that the recombinant protein preparation is endotoxin-free to avoid confounding inflammatory responses.

How can researchers address the challenge of studying MGF 110-1L in the context of the natural host?

Studying MGF 110-1L in the natural porcine host presents several challenges:

• Ethical and biosafety considerations:

  • Solution: Develop ex vivo systems using primary porcine cells and tissues that better recapitulate in vivo conditions.

  • Solution: Establish collaborations with specialized high-containment facilities for essential in vivo work.

• Genetic manipulation limitations:

  • Solution: Use CRISPR/Cas9 technology to modify host factors in porcine cells to test interactions with MGF 110-1L.

  • Solution: Develop porcine cell lines stably expressing or lacking potential MGF 110-1L interaction partners.

• Complex host immune responses:

  • Solution: Use systems biology approaches to analyze global changes in host response when MGF 110-1L is present or absent.

  • Solution: Develop organoid or tissue culture systems that better represent the complexity of host tissues.

• Limited availability of porcine-specific reagents:

  • Solution: Generate custom antibodies against porcine proteins of interest.

  • Solution: Adapt and validate reagents developed for human or mouse systems for use in porcine systems.

What are the most promising approaches to uncover the potential redundant functions among MGF110 family members?

Given the apparent functional redundancy suggested by the lack of phenotype in MGF 110-1L deletion mutants, researchers should consider:

• Multiple gene deletions: Create combinatorial deletions of different MGF110 family members to identify functional overlap, as suggested by preliminary studies with the 1L-5-6L MGF110 deletion mutant .

• Comparative structural analysis: Perform detailed structural comparisons of MGF110 family proteins to identify conserved domains that might mediate similar functions.

• Transcriptomics and proteomics: Compare host cell responses to individual versus multiple MGF110 protein expressions to identify common pathways affected.

• Interactome mapping: Conduct systematic protein-protein interaction studies for all MGF110 family members to identify shared cellular targets.

• Cross-complementation experiments: Test whether expressing other MGF110 proteins can rescue phenotypes associated with MGF 110-1L deletion in specific contexts.

How might studies of MGF 110-1L contribute to ASFV vaccine development?

Understanding MGF 110-1L function has several potential implications for ASFV vaccine development:

• Attenuated vaccine candidates: While MGF 110-1L deletion alone does not attenuate the virus, combining it with other mutations might create optimally balanced attenuated vaccine candidates.

• Immunological insights: Determining if MGF 110-1L modulates host immune responses could inform strategies to enhance vaccine immunogenicity.

• DIVA capability: Deletion mutants could serve as differentiation of infected from vaccinated animals (DIVA) vaccine candidates, where the absence of antibodies to MGF 110-1L could distinguish vaccinated from naturally infected animals.

• Subunit vaccine components: If MGF 110-1L proves immunogenic, it could potentially be included in subunit vaccine formulations targeting multiple viral antigens.

• Host-pathogen interaction targets: Identifying host factors targeted by MGF 110-1L could reveal new pathways for therapeutic intervention or vaccine adjuvant development.

What novel technologies would advance our understanding of MGF 110-1L function?

Several cutting-edge technologies could significantly advance MGF 110-1L research:

• Cryo-electron microscopy: Determine the high-resolution structure of MGF 110-1L alone and in complex with potential binding partners.

• Single-cell analysis: Apply single-cell RNA-seq and proteomics to identify cell-specific responses to MGF 110-1L expression.

• CRISPR screening: Conduct genome-wide CRISPR screens in porcine cells to identify host factors whose loss mimics or enhances MGF 110-1L-associated phenotypes.

• Proximity labeling proteomics: Use BioID or APEX2 fusions to MGF 110-1L to identify proteins in close proximity during infection, revealing potential interaction networks.

• In situ structural biology: Apply techniques like MINFLUX super-resolution microscopy to visualize MGF 110-1L localization and dynamics during viral infection with nanometer precision.

• Deep mutational scanning: Systematically mutate each residue in MGF 110-1L and assess functional consequences to identify critical domains and residues.

• Humanized porcine models: Develop advanced animal models that better recapitulate human-relevant aspects of ASFV infection and immune response.

How can recombinant MGF 110-1L protein be used as a tool in ASFV diagnostic development?

While commercial considerations are avoided, the research applications for diagnostics include:

• Reference standard: Purified recombinant MGF 110-1L can serve as a positive control in research-focused diagnostic assays being developed.

• Antibody development: Generate and characterize monoclonal or polyclonal antibodies against MGF 110-1L for research applications in immunohistochemistry, ELISA, or Western blot.

• Epitope mapping: Identify immunodominant epitopes in MGF 110-1L that could be targeted in more specific diagnostic assays.

• Cross-reactivity studies: Assess potential cross-reactivity between antibodies against MGF 110-1L and proteins from other porcine pathogens to evaluate diagnostic specificity.

• Serosurveillance research: Evaluate the presence and persistence of anti-MGF 110-1L antibodies in experimental infections to determine their utility as infection markers.

What experimental designs would best evaluate the potential role of MGF 110-1L in host range determination?

To investigate MGF 110-1L's potential role in ASFV host range:

• Comparative binding studies: Test MGF 110-1L binding to homologous host factors from different species (domestic pigs, wild boars, warthogs, bushpigs) to identify species-specific interactions.

• Ex vivo replication experiments: Compare replication efficiency of wild-type and MGF 110-1L deletion mutants in primary macrophages from different host species.

• Functional complementation: Express MGF 110-1L variants from ASFV isolates with different host ranges in a common genetic background to assess their impact on replication in cells from various hosts.

• Chimeric protein analysis: Create chimeric proteins combining domains from MGF 110-1L variants associated with different host ranges to map host-specific functional domains.

• Host factor expression patterns: Compare expression levels of putative MGF 110-1L interaction partners across potential host species to identify correlations with susceptibility.

What considerations are important when designing experiments to study MGF 110-1L in the context of ASFV evolution?

When studying MGF 110-1L in an evolutionary context:

• Sequence conservation analysis: Compare MGF 110-1L sequences across historical and contemporary ASFV isolates to identify conserved versus variable regions.

• Selection pressure mapping: Use computational methods to identify sites under positive or negative selection within MGF 110-1L.

• Functional comparison of variants: Express and characterize MGF 110-1L proteins from genetically diverse ASFV isolates to assess functional conservation.

• Recombination analysis: Investigate evidence of recombination events involving MGF 110-1L and other MGF genes throughout ASFV evolution.

• Experimental evolution: Passage ASFV under different selective pressures and monitor changes in the MGF 110-1L sequence and function.

• Cross-species comparison: Compare MGF 110-1L with homologs in related viruses to understand the evolutionary history of this gene family.

• Ancestral sequence reconstruction: Reconstruct and functionally characterize predicted ancestral MGF 110-1L sequences to understand the evolution of its function.