Recombinant African swine fever virus Protein MGF 505-4R (BA71R-027), partial

What are the MGF proteins in African swine fever virus and what is their significance in viral pathogenicity?

MGF (multigene family) proteins in ASFV are encoded by groups of related genes that play critical roles in virus-host interactions, particularly in immune evasion. These include MGF360 and MGF505 families, which contain several members that inhibit host defense pathways. MGF proteins are significant virulence factors that suppress host innate immune responses, allowing ASFV to effectively replicate and cause disease.

How does MGF 505-4R differ structurally and functionally from other MGF family proteins like MGF-505-7R?

While the search results provide limited specific information about MGF 505-4R (BA71R-027) functional characteristics, we can infer its relationship to other MGF505 family proteins. MGF 505-4R belongs to the same multigene family as the well-characterized MGF-505-7R, suggesting possible shared structural features and immune evasion functions.

The MGF-505-7R protein has been extensively studied and demonstrated to:

• Inhibit JAK-STAT1 signaling by interacting with JAK1/JAK2

• Suppress cGAS-STING signaling pathway

• Inhibit IL-1β and type I IFN production

• Interact with IRF9 and inhibit ISGF3 nuclear translocation

By comparison, MGF 505-4R likely has its own unique role in virus-host interactions, possibly targeting different components of immune signaling pathways. Further comparative research between these proteins would be valuable for understanding the diverse strategies ASFV employs to evade host immunity.

What expression systems are recommended for producing recombinant MGF 505-4R protein for laboratory research?

Recombinant MGF 505-4R protein can be produced in multiple expression systems, each with distinct advantages depending on your research needs:

• E. coli expression system: Suitable for basic structural studies and producing large quantities of protein. Available with product codes like CSB-EP805662AEJ or as biotinylated versions (CSB-EP805662AEJ-B) .

• Yeast expression system: Offers improved protein folding and some post-translational modifications compared to bacterial systems. Available with product code CSB-YP805662AEJ .

• Baculovirus expression system: Provides more extensive eukaryotic post-translational modifications and better protein folding. Available with product code CSB-BP805662AEJ .

• Mammalian cell expression system: Offers the most authentic post-translational modifications and protein folding, ideal for functional studies. Available with product code CSB-MP805662AEJ .

The optimal choice depends on your specific experimental requirements. For studies focusing on protein-protein interactions or functional assays, mammalian or baculovirus systems are recommended due to their superior post-translational modifications. For structural studies or applications requiring large quantities of protein, E. coli or yeast systems may be more cost-effective.

How can recombinant MGF 505-4R be used in experimental studies of ASFV immune evasion mechanisms?

Recombinant MGF 505-4R can be utilized in multiple experimental approaches to study ASFV immune evasion:

• Protein-protein interaction studies: Use co-immunoprecipitation assays with tagged recombinant MGF 505-4R to identify host cell binding partners, similar to studies with MGF-505-7R that revealed interactions with JAK1/JAK2 and IRF9 .

• Reporter gene assays: Employ luciferase reporter systems to evaluate MGF 505-4R's effects on signaling pathways like JAK-STAT, NF-κB, or ISRE promoter activity. This approach revealed that MGF-505-7R inhibits IFN-β and IL-1β signaling .

• Immunological assays: Analyze the impact of MGF 505-4R on cytokine production using ELISA or flow cytometry in relevant cell models (porcine alveolar macrophages).

• Confocal microscopy: Examine the subcellular localization of MGF 505-4R and potential colocalization with host factors, as demonstrated with MGF-505-7R and STING .

• Comparative studies: Compare MGF 505-4R with other MGF proteins to identify shared and unique functions in immune evasion, providing insights into ASFV's multifaceted approach to host manipulation.

These approaches would help elucidate MGF 505-4R's specific role in ASFV pathogenesis and potentially identify new targets for vaccine development.

What experimental controls should be included when studying the immunomodulatory effects of recombinant MGF 505-4R?

When studying immunomodulatory effects of recombinant MGF 505-4R, include these essential controls:

• Empty vector control: Cells transfected with the expression vector lacking the MGF 505-4R gene to account for vector-induced effects.

• Irrelevant viral protein control: A non-immunomodulatory viral protein expressed under identical conditions to distinguish MGF 505-4R-specific effects from general viral protein expression effects.

• Known immunomodulatory protein control: Include MGF-505-7R or another well-characterized immunomodulatory protein as a positive control for assay validation.

• Dose-dependent response: Test multiple concentrations of MGF 505-4R to establish dose-response relationships.

• Time-course experiments: Analyze effects at different time points to capture both immediate and delayed responses.

• Cell-type specificity controls: Test in both relevant cell types (porcine alveolar macrophages) and control cell lines to determine cell type specificity.

• Pathway-specific positive controls: Include pathway-specific activators (e.g., IFN-γ for JAK-STAT pathway, cGAMP for STING pathway) to validate assay functionality.

• Biological replicates: Perform at least three independent experiments to ensure reproducibility.

• Pathway inhibitor controls: Use specific inhibitors of relevant pathways to confirm the specificity of observed effects.

These controls will help distinguish true MGF 505-4R immunomodulatory effects from experimental artifacts and provide robust data for publication.

How do the immunomodulatory mechanisms of MGF 505-4R compare with other MGF proteins like MGF-505-7R in ASFV pathogenesis?

While specific information about MGF 505-4R mechanisms is limited in the search results, we can compare it with the well-characterized MGF-505-7R to develop research hypotheses:

MGF-505-7R employs multiple mechanisms to suppress host immunity:

• JAK-STAT pathway inhibition: MGF-505-7R interacts with JAK1 and JAK2, promoting their degradation by upregulating E3 ubiquitin ligase RNF125 expression and inhibiting Hes5 expression . This suppresses IFN-γ-mediated signaling.

• cGAS-STING pathway suppression: MGF-505-7R interacts with STING and promotes its degradation via the autophagy pathway by enhancing ULK1 expression . This inhibits type I IFN production.

• IFN-I signaling inhibition: MGF-505-7R interacts with IRF9 and inhibits ISGF3 heterotrimer formation and nuclear translocation, suppressing IFN-stimulated gene expression .

• Inflammasome inhibition: MGF-505-7R interacts with IKKα in the IKK complex to inhibit NF-κB activation and binds to NLRP3 to inhibit inflammasome formation, reducing IL-1β production .

MGF 505-4R likely targets different components of these pathways or entirely different mechanisms. Research comparing deletion mutants of both proteins and their effects on different signaling pathways would be valuable for understanding the complementary roles these proteins play in immune evasion.

What is the potential of MGF 505-4R deletion mutants for developing attenuated ASFV vaccines compared to other gene deletion approaches?

Based on research with related MGF proteins, MGF 505-4R deletion mutants could have significant potential for attenuated vaccine development. The comparative effectiveness of different deletion approaches can be evaluated:

• MGF-505-7R deletion: Deletion of MGF-505-7R highly attenuates ASFV virulence in pigs . MGF-505-7R-deficient ASFV induces higher levels of IL-1β and IFN-β production and is more susceptible to IFN-β . This suggests MGF-505-7R deletion is a promising approach for live attenuated vaccines.

• CD2v (EP402R) deletion: The BA71ΔCD2 strain (deletion of viral CD2v gene) is attenuated in vivo and provides protection against both homologous (BA71) and heterologous (E75, Georgia 2007/1) ASFV strains . This cross-protection correlates with the ability to induce specific CD8+ T cells.

• Multiple MGF deletions: Deletion of MGF360 (10L, 11L, 12L, 13L, 14L) and MGF505 (1R, 2R, 3R) genes results in reduced virus growth in vitro and attenuation in vivo .

• Combination approaches: Double deletion mutants (BA71∆CD2DP96R and BA71∆CD2EP153R) were studied, but unexpectedly showed decreased vaccine efficacy compared to the single mutant BA71∆CD2 .

For MGF 505-4R, research should investigate:

• Its specific contribution to virulence

• Whether its deletion attenuates the virus sufficiently alone

• How it combines with other deletions (like MGF-505-7R)

• The immune response elicited by MGF 505-4R deletion mutants

• Protection efficacy against homologous and heterologous challenges

The optimal approach likely involves a carefully balanced deletion strategy that sufficiently attenuates the virus while maintaining immunogenicity.

What are the key experimental challenges in characterizing the structure-function relationship of MGF 505-4R protein?

Characterizing the structure-function relationship of MGF 505-4R presents several key experimental challenges:

• Protein expression and purification: MGF proteins often contain hydrophobic regions that may affect solubility and stability. Expression in different systems (bacterial, yeast, insect, mammalian) may yield proteins with varying degrees of functionality due to post-translational modifications .

• Structural determination: The lack of homology to known proteins makes structural prediction difficult. X-ray crystallography or cryo-EM studies are challenging due to potential flexibility or membrane association of the protein.

• Functional domain mapping: Identifying functional domains requires systematic truncation studies similar to those performed with MGF-505-7R, which revealed that the first transmembrane domain (1-175 aa) was required for STING interaction .

• Host protein interaction network: MGF proteins likely interact with multiple host proteins across different signaling pathways. Comprehensive interactome studies using techniques like BioID or IP-MS in porcine cells are challenging but necessary.

• Relevance to different ASFV strains: Genetic variability between ASFV isolates may affect MGF 505-4R function. Comparative studies across strains are needed but complicated by biosafety requirements.

• In vivo validation: Testing structure-function hypotheses in the natural host (pigs) is expensive and requires specialized facilities, limiting the throughput of validation studies.

• Temporal dynamics: The timing of MGF 505-4R expression and its effects during infection add another layer of complexity requiring time-course experiments in infected cells.

Addressing these challenges requires a multidisciplinary approach combining structural biology, molecular virology, immunology, and in vivo studies.

What are the optimal methods for evaluating the effects of recombinant MGF 505-4R on JAK-STAT and other immune signaling pathways?

To evaluate MGF 505-4R effects on immune signaling pathways, consider these methodological approaches:

• JAK-STAT pathway analysis:

  • Phosphorylation assays: Examine STAT1/2 phosphorylation using phospho-specific antibodies via Western blotting after IFN stimulation in the presence/absence of MGF 505-4R .

  • Nuclear translocation assays: Use nucleocytoplasmic fractionation or confocal microscopy to assess STAT1/2 nuclear translocation .

  • Luciferase reporter assays: Measure GAS (IFN-γ) or ISRE (IFN-α/β) promoter activity using dual luciferase systems .

  • qRT-PCR for ISGs: Quantify expression of ISGs (ISG15, ISG54, MxA, IRF1, CXCL9, GBP1) in response to IFN stimulation .

• cGAS-STING pathway analysis:

  • STING dimerization assays: Evaluate STING dimerization by non-reducing SDS-PAGE after cGAMP stimulation .

  • STING degradation assays: Monitor STING protein levels over time in the presence of MGF 505-4R, with autophagy inhibitors (3-MA) to determine degradation mechanisms .

  • Co-localization studies: Use confocal microscopy to assess MGF 505-4R and STING co-localization .

• NF-κB and inflammasome pathway analysis:

  • NF-κB reporter assays: Measure NF-κB promoter activity using luciferase reporters .

  • IL-1β ELISA: Quantify mature IL-1β secretion after inflammasome activation .

  • ASC speck formation: Visualize inflammasome assembly by microscopy of ASC specks .

• Protein-protein interaction studies:

  • Co-immunoprecipitation: Use tagged versions of MGF 505-4R to identify binding partners .

  • GST pull-down assays: Employ recombinant proteins to confirm direct interactions.

  • Domain mapping: Create truncation mutants to identify interaction domains .

Use porcine alveolar macrophages (PAMs) or relevant porcine cell lines whenever possible for physiological relevance, and include appropriate controls as outlined in question 2.2.

What techniques are most effective for studying the role of MGF 505-4R in ASFV replication and virulence in cell culture and animal models?

For studying MGF 505-4R's role in ASFV replication and virulence, employ these techniques:

Cell Culture Studies:

• Construction of gene deletion mutants:

  • Homologous recombination to generate MGF 505-4R-deleted ASFV, using fluorescent markers for virus purification .

  • PCR verification of gene deletion and whole-genome sequencing to confirm no off-target mutations .

• Virus replication kinetics:

  • Multistep growth curves in porcine alveolar macrophages (PAMs) .

  • Quantification of viral DNA by qPCR targeting conserved regions like p72 (B646L) or p30 (CP204L) .

  • Assessment of virus protein production by Western blotting or immunofluorescence .

• Comparative studies with wild-type virus:

  • Side-by-side infection experiments at various MOIs (0.01, 0.1, 1) .

  • Analysis of virus sensitivity to IFN-I/II treatment by pretreating cells before infection .

• Host response analysis:

  • qRT-PCR for cytokine and ISG expression (IFN-β, IL-1β, ISG15, MxA) .

  • Multiplex cytokine assays on culture supernatants.

  • Pathway-specific reporter assays comparing wild-type and MGF 505-4R-deleted virus .

Animal Model Studies:

• Infection model design:

  • Group size considerations (minimum 6 pigs per group for statistical power) .

  • Inoculation route optimization (intramuscular is commonly used) .

  • Dose determination (typically 10-10^4 HAD50 depending on virulence) .

• Clinical and virological monitoring:

  • Daily clinical scoring (temperature, clinical signs) .

  • Blood sampling schedule for viremia quantification by qPCR .

  • Cytokine profiling in serum samples .

• Immune response characterization:

  • ASFV-specific antibody ELISA for humoral response .

  • ELISpot or intracellular cytokine staining for T-cell responses .

  • Immunophenotyping of peripheral blood mononuclear cells.

• Pathological examination:

  • Tissue collection from multiple organs for viral load quantification .

  • Histopathological examination with immunohistochemistry.

  • Comparison of lesion severity between wild-type and deletion mutant viruses.

These methodologies should be tailored to the specific research question, with appropriate biosafety measures for handling ASFV in laboratory and animal settings.

What are the recommended protocols for measuring the impact of MGF 505-4R on various aspects of host immune response in porcine cell models?

For measuring MGF 505-4R's impact on host immune responses in porcine cells, employ these specialized protocols:

• Innate immune signaling assessment in porcine alveolar macrophages (PAMs):

  a) Type I IFN pathway:

  • IFN-β mRNA quantification: Extract RNA using TRIzol and perform RT-qPCR with porcine-specific primers for IFN-β and housekeeping genes (GAPDH, β-actin) .

  • IFN-β protein measurement: Use porcine-specific ELISA kits to quantify secreted IFN-β in culture supernatants .

  • ISG expression analysis: Measure ISG15, ISG54, MxA, and Mx1 expression by RT-qPCR following stimulation with poly(dA:dT) or infection .

  b) JAK-STAT pathway:

  • STAT1/2 phosphorylation: After IFN stimulation, lyse cells, perform SDS-PAGE, and Western blot using phospho-specific antibodies against porcine STAT1 (Y701) and STAT2 (Y690) .

  • Nuclear translocation: Perform nuclear-cytoplasmic fractionation followed by Western blot analysis of ISGF3 components (STAT1, STAT2, IRF9) .

  • Target gene induction: Measure CXCL9, GBP1, IRF1 expression by RT-qPCR after IFN-γ stimulation .

• Inflammasome activation in PAMs:

  a) IL-1β production measurement:

  • mRNA level: RT-qPCR for pro-IL-1β expression .

  • Protein level: ELISA for mature IL-1β in supernatants after LPS priming and inflammasome activation .

  • Western blot: Detection of pro-IL-1β and mature IL-1β (p17) in cell lysates and supernatants .

  b) Inflammasome assembly visualization:

  • Immunofluorescence microscopy: Stain for NLRP3, ASC, and caspase-1 to visualize inflammasome formation .

  • ASC oligomerization: Perform ASC oligomer cross-linking assay and Western blot analysis .

• Protein-protein interaction studies in porcine cells:

  a) Co-immunoprecipitation:

  • Transfect PAMs with tagged MGF 505-4R constructs using nucleofection.

  • Immunoprecipitate with tag-specific antibodies and blot for suspected interaction partners (JAK1, JAK2, STING, IRF9, IKKα, NLRP3) .

  b) Proximity ligation assay (PLA):

  • Visualize protein interactions in situ using antibodies against MGF 505-4R and target proteins .

• Comparative virus infection studies:

  a) Side-by-side infection:

  • Infect PAMs with wild-type and MGF 505-4R-deleted ASFV at MOI 0.1 .

  • Compare viral replication (qPCR, Western blot) .

  • Analyze immune response kinetics at multiple time points (6, 12, 24, 48 hours) .

  b) IFN sensitivity assay:

  • Pre-treat PAMs with increasing concentrations of porcine IFN-β.

  • Infect with wild-type and MGF 505-4R-deleted ASFV.

  • Quantify viral replication by qPCR and immunofluorescence .

These protocols should be adapted to available resources and specific research questions, with emphasis on appropriate controls and statistical analysis for robust interpretation of results.

What are the key considerations for designing gene deletion experiments to study MGF 505-4R function in the context of ASFV infection?

When designing gene deletion experiments to study MGF 505-4R function in ASFV infection, consider these critical factors:

• Deletion strategy design:

  • Target sequence selection: Design precise deletion boundaries to avoid disrupting overlapping genes or regulatory elements.

  • Marker gene selection: Consider using fluorescent markers (GFP, mCherry) for easy visualization or selectable markers for enrichment.

  • Homologous flanking regions: Include 500-1000 bp homologous sequences flanking the target gene to ensure efficient recombination .

  • Verification strategy: Design PCR primers outside the homologous regions to confirm successful deletion .

• Recombinant virus generation:

  • Cell system selection: Use permissive cells capable of supporting ASFV growth (PAMs or adapted cell lines).

  • Transfection-infection approach: Transfect cells with the deletion construct and subsequently infect with wild-type ASFV .

  • Virus purification: Employ limited dilution or plaque purification methods for multiple rounds (≥10) to ensure genetic homogeneity .

  • Verification methods: Use PCR, restriction enzyme analysis, and whole-genome sequencing to confirm the correct deletion and absence of unwanted mutations .

• Control virus construction:

  • Revertant virus: Create a revertant virus by re-introducing the deleted gene to confirm phenotype specificity.

  • Reporter control virus: Generate a control virus with the same marker but without deleting MGF 505-4R.

  • Multi-gene consideration: Consider creating viruses with deletion of other MGF genes (e.g., MGF-505-7R) for comparative analysis .

• Phenotypic characterization:

  • Growth kinetics: Compare replication efficiency in PAMs at various MOIs (0.01-10) .

  • Host response analysis: Evaluate effects on type I IFN production, JAK-STAT signaling, and inflammatory responses .

  • Differential susceptibility: Test sensitivity to IFN-β pre-treatment to identify immune evasion functions .

  • Protein-protein interactions: Investigate whether host protein interactions change in the context of infection versus transfection .

• In vivo studies considerations:

  • Virus dose standardization: Use HAD50 or TCID50 determinations for accurate dosing .

  • Clinical scoring system: Develop a comprehensive clinical scoring system for ASFV-specific symptoms .

  • Sampling schedule: Design a detailed timeline for blood and tissue collection to capture key events in viral pathogenesis .

  • Ethical considerations: Follow the 3Rs principle (replacement, reduction, refinement) and obtain proper ethical approvals.

• Experimental challenges:

  • Biosafety requirements: Work with ASFV requires appropriate biosafety measures (BSL-3 for virulent strains).

  • Genetic stability: Monitor the stability of the deletion through multiple passages.

  • Compensatory mutations: Be alert to potential compensatory mutations that might emerge during virus propagation.

  • Pleiotropic effects: Consider that deletion of one gene might affect the expression or function of other viral genes.

By carefully addressing these considerations, researchers can design robust experiments to elucidate the specific functions of MGF 505-4R in ASFV pathogenesis and immune evasion.

How might comparative analysis of MGF 505-4R across different ASFV genotypes inform vaccine development strategies?

Comparative analysis of MGF 505-4R across ASFV genotypes could significantly advance vaccine development through several key mechanisms:

• Conservation assessment and variant identification:

  • Analysis of MGF 505-4R sequence conservation across genotypes I and II strains could reveal invariant regions as potential targets for broadly protective vaccines .

  • Identification of genotype-specific variations might explain differences in virulence and host range. For example, search result mentions unique genetic markers in C962R, I329L, and MGF 505-11L genes distinguishing Vietnamese recombinants from Chinese strains.

• Structure-function correlations:

  • Mapping sequence variations to functional domains could identify critical regions for immune evasion that should be modified in attenuated vaccine candidates.

  • Comparing MGF 505-4R with the well-characterized MGF-505-7R could reveal shared functional domains critical for virulence .

• Cross-protection potential evaluation:

  • Understanding MGF 505-4R conservation could help predict whether deletion mutants might provide cross-protection against multiple genotypes, similar to how BA71ΔCD2 protects against both genotype I strains (BA71, E75) and genotype II (Georgia 2007/1) .

• Optimal attenuation strategies:

  • Comparing the contributions of different MGF genes to virulence across genotypes could inform whether MGF 505-4R deletion should be combined with other modifications.

  • Results from studies with combinations of deletions (MGF360 and MGF505) suggest that certain combinations are more effective for attenuation than others .

• Recombination hotspot identification:

  • Analysis of naturally occurring recombinant strains containing MGF 505-4R variants could identify recombination-prone regions and inform strategies to enhance genetic stability of vaccine candidates .

This comprehensive approach would help develop rationally designed ASFV vaccines with optimal attenuation, cross-protection potential, and genetic stability across different epidemiological contexts.

What are the most promising research directions for developing MGF 505-4R-targeted antiviral strategies against ASFV?

Several promising research directions for MGF 505-4R-targeted antiviral strategies warrant investigation:

• Small molecule inhibitors:

  • Structure-based drug design targeting critical functional domains of MGF 505-4R.

  • High-throughput screening for compounds that disrupt MGF 505-4R interactions with host proteins.

  • Repurposing of existing drugs that target similar viral immune evasion mechanisms.

• Peptide-based inhibitors:

  • Development of peptide mimetics that compete with MGF 505-4R for binding to its host targets.

  • Cell-penetrating peptides designed to disrupt MGF 505-4R function inside infected cells.

• Rational vaccine design:

  • Generation of MGF 505-4R deletion mutants alone or in combination with other attenuating mutations.

  • Comparative studies with MGF-505-7R deletion mutants that have shown promise as vaccine candidates .

  • Design of chimeric viruses where MGF 505-4R is replaced with modified versions lacking immune evasion functions but retaining immunogenicity.

• Host-directed therapeutics:

  • Targeting host pathways exploited by MGF 505-4R to restore immune responses.

  • If MGF 505-4R functions similarly to MGF-505-7R, strategies could include:

    • JAK-STAT pathway enhancers to counteract possible JAK1/JAK2 degradation .

    • STING pathway stabilizers to overcome potential cGAS-STING inhibition .

    • Anti-autophagy compounds if MGF 505-4R promotes autophagy-dependent degradation of immune factors .

• CRISPR-Cas strategies:

  • Development of CRISPR-Cas systems targeting the MGF 505-4R gene to disrupt its expression during infection.

  • Creation of genetically modified pigs with enhanced resistance to MGF 505-4R immune evasion strategies.

• Combination approaches:

  • Synergistic targeting of multiple MGF proteins simultaneously to overcome potential functional redundancy.

  • Combining MGF 505-4R inhibitors with conventional antivirals that target viral replication.

• Cross-protective immunity induction:

  • Design of immunogens based on conserved epitopes from MGF 505-4R and related proteins.

  • Development of heterologous prime-boost vaccination approaches incorporating MGF 505-4R antigens.

These approaches should be pursued in parallel, with initial in vitro screening followed by validation in porcine cell models and eventually in vivo testing in appropriate animal models.