Recombinant African swine fever virus Major structural protein p17 (War-117)
Description
Introduction to African Swine Fever Virus and p17 Protein
African swine fever virus (ASFV) is a highly pathogenic large DNA virus responsible for African swine fever (ASF), a severe disease affecting domestic pigs and wild boars. The disease has caused significant economic losses in the global swine industry, particularly following its introduction to China in 2018 and subsequent spread across Asia and parts of Europe . ASFV encodes more than 150 proteins that establish complex interactions with the host, enabling the virus to evade immune defenses and successfully replicate within host cells .
The p17 protein, encoded by the D117L gene, is a major structural transmembrane protein located in the capsid and inner lipid envelope of ASFV . This protein plays a critical role in the viral architecture and is essential for the assembly and maturation of the icosahedral capsid as well as general virus viability . The gene encoding p17 was mapped and sequenced in 1995, revealing its position in the EcoRI D fragment of the viral genome with an open reading frame designated D117R . Transcriptional analysis demonstrated that the p17 gene is expressed late in the viral infection cycle, consistent with its structural role in virion assembly .
Genomic Location and Expression
The p17 gene (D117L) is located in the 140-150 kb central region of the ASFV genome, close to the right variable region . The gene encodes a protein of 117 amino acids with a deduced molecular mass of approximately 13,000 Da, though it typically appears as a 17 kDa protein in gel electrophoresis, hence its designation as p17 . As a late-expressed viral gene, p17 production coincides with the assembly phase of the viral replication cycle, emphasizing its importance in virion construction rather than early replication events .
Molecular Properties and Domains
The p17 protein contains important functional domains that contribute to its various biological activities. A particularly significant region is the transmembrane domain, spanning amino acids 39-59, which is required for its interaction with Stimulator of Interferon Genes (STING) and subsequent inhibition of the cGAS-STING pathway . This domain likely facilitates p17's localization to cellular membranes, particularly the endoplasmic reticulum (ER) and Golgi apparatus, where it can interfere with host signaling pathways .
In addition, monoclonal antibody studies have identified a conservative linear epitope within the N-terminal region of p17, specifically at amino acids 3-11 (TETSPLLSH) . This conserved region represents an important immunogenic site that can be targeted for diagnostic purposes and potentially for vaccine development. The identification of conserved epitopes is particularly valuable given the genetic diversity observed among ASFV isolates .
Role in Viral Assembly and Maturation
P17 plays a critical structural role in ASFV assembly. It is essential for the progression of viral membrane precursors toward icosahedral intermediates during virion formation . The protein binds to the capsid protein p72 and closely connects the inner membrane to the outer shell of the ASFV virus, essentially serving as a molecular bridge that helps maintain the structural integrity of the virion . The trimeric organization of p17 at the interfaces of capsomers provides additional stability to the viral structure, which is crucial for producing viable viral particles capable of infecting new host cells .
Modulation of Cell Proliferation and Cell Cycle
Research has demonstrated that p17 significantly impacts host cell functions beyond its structural role in the virus. Studies examining the effects of p17 on cell proliferation revealed that this viral protein reduces cell proliferation by causing cell cycle arrest at the G2/M phase . This effect was observed in multiple cell types including 293T, PK15, and primary porcine alveolar macrophages (PAM) cells, suggesting a conserved mechanism of action across different host cell types .
The cell cycle arrest induced by p17 represents a significant modulation of host cell physiology that may benefit viral replication. By halting cell division at the G2/M checkpoint, the virus potentially creates a cellular environment more conducive to viral protein production and assembly. This cell cycle disruption is part of a complex cascade of cellular responses triggered by p17 expression, which includes oxidative stress and endoplasmic reticulum stress .
Induction of Oxidative Stress and ROS Production
A key finding regarding p17's cellular effects is its ability to induce oxidative stress and increase the levels of intracellular reactive oxygen species (ROS) . Importantly, experimental manipulation of ROS levels revealed a functional connection between ROS production and cell cycle arrest. When researchers decreased the level of ROS in p17-expressing cells, they observed a partial reversal of the cell cycle arrest and prevention of the decrease in cell proliferation induced by the p17 protein . This suggests that ROS elevation is a crucial mediator of p17's effects on cell cycle progression.
These findings establish a mechanistic pathway through which p17 disrupts normal cellular functions: p17 expression → increased ROS production → cell cycle arrest → reduced cell proliferation. This cascade may contribute to the cytopathic effects observed during ASFV infection and potentially plays a role in the pathogenesis of African swine fever .
Endoplasmic Reticulum Stress Response
Beyond oxidative stress, p17 also induces endoplasmic reticulum (ER) stress in host cells . The ER is a critical organelle involved in protein folding, modification, and quality control. When the ER's capacity to properly fold proteins is compromised, cells activate the unfolded protein response (UPR), a mechanism aimed at restoring ER homeostasis or triggering apoptosis if the stress cannot be resolved.
Experimental evidence has shown that alleviating ER stress in p17-expressing cells leads to decreased production of ROS and prevents the reduction in cell proliferation induced by p17 . This finding establishes a sequential relationship where ER stress appears to occur upstream of ROS production in the p17-induced cellular response pathway. The complete pathway can thus be expanded to: p17 expression → ER stress → increased ROS production → cell cycle arrest → reduced cell proliferation.
Immune Evasion Through Inhibition of cGAS-STING Pathway
Perhaps one of the most significant functional aspects of p17 in terms of viral pathogenesis is its ability to interfere with host innate immune responses. Research has demonstrated that p17 exerts a negative regulatory effect on the cGAS-STING signaling pathway, which is a crucial component of the innate immune response to viral infections .
The cGAS-STING pathway recognizes cytosolic DNA (often from invading pathogens) and triggers the production of type I interferons and other proinflammatory cytokines. Through inhibition of this pathway, p17 helps ASFV evade host immune detection and response. Mechanistically, p17 localizes to the ER and Golgi apparatus where it physically interacts with STING, a key adaptor protein in the signaling cascade . This interaction interferes with STING's ability to recruit downstream signaling molecules TBK1 and IKKε, effectively blocking signal transduction and the subsequent activation of immune responses .
The transmembrane domain (amino acids 39-59) of p17 has been identified as crucial for this interaction with STING and the resulting inhibition of the cGAS-STING pathway . Experimental evidence using p17-specific siRNA showed that knocking down p17 during ASFV infection led to upregulation of IFN-β, ISG15, ISG56, IL-6, and IL-8 gene transcriptions in ASFV-infected primary porcine alveolar macrophages, confirming p17's role in suppressing these immune response genes during normal viral infection .
Expression Systems for p17 Production
Successful expression of recombinant p17 has been achieved using different host systems, providing valuable tools for research and diagnostic applications. The protein has been successfully produced in bacterial systems such as E. coli, where it can be expressed with various tags (such as His-tag) to facilitate purification . Creative BioMart offers recombinant full-length p17 protein produced in E. coli with a His-tag, demonstrating the commercial availability of this research tool .
Beyond bacterial expression, mammalian cell systems have also been employed. Chinese Hamster Ovary (CHO) cells using a suspension culture system have successfully expressed p17, offering advantages for certain applications requiring eukaryotic post-translational modifications . In these expression systems, the levels of p17 production were observed to increase gradually with transfection time, reaching peak expression at 5 days post-transfection .
The expressed recombinant protein maintains important biological properties, including reactivity with ASFV-positive serum, confirming its antigenic relevance and potential use in diagnostic and immunological studies .
Development of Diagnostic Tools
The availability of recombinant p17 has enabled the development of valuable diagnostic tools for ASFV detection. An indirect enzyme-linked immunosorbent assay (ELISA) based on purified p17 has been established and optimized for detecting ASFV antibodies in clinical samples . This ELISA method has demonstrated effectiveness in detecting clinical ASFV infections and monitoring antibody levels following vaccination with recombinant viral vectors expressing p17 .
The optimal conditions for this p17-based ELISA have been determined as follows:
| Parameter | Optimized Condition |
|---|---|
| p17 concentration | 400 ng/well |
| Serum dilution | 1:100 |
| Coating solution | Phosphate solution at 4°C overnight |
| Blocking solution | 5% BSA at 37°C for 1 hour |
| Secondary antibody dilution | 1:20,000 |
| Serum incubation time | 45 minutes |
| Secondary antibody incubation | 45 minutes |
| Color development conditions | TMB for 15 minutes at 37°C |
These optimized conditions ensure reliable detection of ASFV antibodies, making this assay a valuable tool for epidemiological surveillance .
Monoclonal Antibodies Against p17
Monoclonal antibodies (mAbs) targeting p17 have been successfully developed and characterized. These antibodies recognize a conservative linear epitope (TETSPLLSH) located at amino acids 3-11 of the p17 protein . The identification of this conserved epitope is significant for several reasons: it indicates a region of the protein that is maintained across different ASFV strains, it represents a potentially immunodominant portion of the protein, and it offers a specific target for diagnostic and research applications.
The development process for these monoclonal antibodies involved immunizing BALB/c mice with purified p17 mixed with colloidal manganese adjuvant, followed by cell fusion and selection of hybridoma cells that secrete specific antibodies against p17 . The resulting monoclonal antibodies exhibited specific reactivity and were conducive to the identification of recombinant vectors expressing p17, such as recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing p17 .
These monoclonal antibodies serve as important tools for ASFV research, potentially enabling antigen detection, immunohistochemistry, and further characterization of p17's roles in viral pathogenesis.
Applications in Vaccine Research
The recombinant p17 protein and tools developed around it offer promising applications in vaccine research against ASFV. The identification of conserved epitopes like the TETSPLLSH sequence provides potential targets for vaccine development. Additionally, the ability to express p17 in various systems allows for its incorporation into different vaccine platforms, including subunit vaccines or viral vector-based approaches .
Research has already demonstrated the use of recombinant viral vectors, specifically recombinant PRRSV expressing p17, as potential vaccine candidates . The p17-based ELISA has been shown to effectively monitor antibody levels following vaccination with these vectors, providing a valuable tool for assessing vaccine-induced immunity .
Furthermore, understanding p17's role in immune evasion through inhibition of the cGAS-STING pathway offers insights into how vaccines might need to be designed to overcome this viral immune evasion strategy. This knowledge could guide the development of more effective ASFV vaccines by potentially including modified versions of p17 that lack immunosuppressive functions while maintaining immunogenicity .
Emerging Roles in ASFV Pathogenesis
The multifunctional nature of p17 continues to be unveiled through ongoing research. Its roles in cell cycle modulation, stress induction, and immune evasion highlight how ASFV has evolved to manipulate host cell functions through individual viral proteins. The connections between these different activities—particularly the pathway from ER stress to ROS production to cell cycle arrest—warrant further investigation to fully understand the molecular mechanisms involved .
The involvement of p17 in ASFV pathogenesis extends beyond its structural role, and further research may reveal additional functions or interactions with host proteins. Understanding these interactions could provide new targets for antiviral development or vaccine strategies. The specific contribution of p17-induced effects to clinical disease manifestations remains to be fully elucidated and represents an important area for future research .
Potential for Therapeutic and Preventive Strategies
The identification of p17's role in immune evasion through STING inhibition presents an opportunity for developing countermeasures that specifically target this interaction. Small molecules or peptides that prevent p17-STING binding could potentially restore the host's ability to mount an effective innate immune response against ASFV infection .
Additionally, the conserved epitopes identified in p17 offer potential targets for broadly protective vaccines. As ASFV continues to spread globally and less virulent strains emerge, there is an increasing need for effective vaccines. The development of recombinant vectors expressing modified p17 or subunit vaccines incorporating p17 epitopes represents promising strategies worth exploring .
Advancing Diagnostic Applications
The successful development of p17-based diagnostic tools demonstrates the protein's value for ASFV detection. Future research could focus on improving the sensitivity and specificity of these assays, perhaps through multiplexing with other ASFV antigens or developing rapid field-deployable test formats .
The monoclonal antibodies against p17 have applications beyond ELISA, including potential use in immunochromatographic assays, fluorescence microscopy for viral localization studies, or affinity purification of viral components. Expanding the toolkit of p17-targeted reagents will continue to facilitate ASFV research and surveillance efforts .
Product Specs
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Target Background
Q&A
What is the structural composition and cellular localization of ASFV p17?
ASFV p17 is a 117-amino acid transmembrane protein encoded by the D117L gene. The protein contains three glycosylation sites at positions N12, N61, and N97, with a single transmembrane domain spanning amino acids 39-59 . Immunofluorescence assays and co-localization studies have demonstrated that p17 primarily localizes to the endoplasmic reticulum (ER) and Golgi apparatus within infected cells . This localization pattern is consistent with its role as an abundant structural protein found in the viral internal envelope . Determining the precise subcellular localization is methodologically important as it provides insight into potential protein-protein interactions and functional roles during viral replication.
How does p17 contribute to ASFV immune evasion mechanisms?
ASFV p17 functions as an immunosuppressive protein that inhibits the cGAS-STING signaling pathway, which is crucial for type I interferon (IFN) production during viral infection . Experimental evidence shows that p17 exerts its inhibitory effect by directly interacting with STING (Stimulator of Interferon Genes) and interfering with STING's ability to recruit downstream signaling proteins TBK1 and IKKε . This interaction prevents the formation of the "STING-IRF3-TBK1" complex required for interferon production . Researchers studying these mechanisms typically employ co-immunoprecipitation assays and immunofluorescence microscopy to detect and visualize these protein-protein interactions in cell culture models.
What experimental approaches are used to study p17 function in ASFV infection?
Several complementary experimental approaches have been employed to investigate p17 function:
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RNA interference: siRNA-mediated knockdown of p17 expression in infected primary porcine alveolar macrophages (PAMs) has been used to assess its role in viral replication and immune evasion .
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Recombinant virus systems: Inducible expression systems have been developed where the p17-encoding D117L gene is placed under the control of a synthetic promoter derived from the late p72 promoter, allowing for controlled expression studies .
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Co-immunoprecipitation assays: These are used to detect physical interactions between p17 and host factors like STING .
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RT-qPCR analysis: This approach quantifies the expression of interferon-stimulated genes and inflammatory cytokines in response to p17 manipulation .
How does the transmembrane domain of p17 mediate its interaction with STING?
The transmembrane domain of p17 (amino acids 39-59) has been identified as essential for its interaction with STING and subsequent inhibition of the cGAS-STING pathway . Recent structural analyses suggest that most of this transmembrane region, except amino acids 39-43, may be critical for the STING interaction . On the STING side, the N-terminal portion and middle cyclic dinucleotide-binding domain appear to be the regions involved in binding p17, while the C-terminal CTT domain is not directly involved .
Methodologically, researchers investigating these structure-function relationships employ:
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Targeted mutagenesis to create domain deletion or point mutation variants
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Protein-protein interaction assays with these variants
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Functional reporter assays to measure downstream signaling activity
Understanding these specific interaction domains could potentially lead to the development of targeted inhibitors that disrupt p17-STING binding without affecting normal STING function.
What are the differences in p17 function between various ASFV genotypes and isolates?
While the core immune evasion function of p17 appears to be conserved across ASFV strains, there may be variations in its efficacy or specific mechanisms between different viral genotypes. Current research has primarily focused on a limited number of ASFV isolates. When comparing p17 sequences and functions across genotypes, researchers should consider:
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Sequence conservation analysis of the D117L gene across different ASFV genotypes
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Comparative functional assays measuring the impact on IFN signaling
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Binding affinity assessments with host STING proteins from different species
These comparative approaches are essential for understanding strain-specific virulence determinants and developing broadly effective control strategies for diverse ASFV isolates.
What are the implications of p17 knockdown for the development of potential ASFV vaccines?
Experiments using p17-specific siRNA have demonstrated that knockdown of p17 expression reduces viral replication and enhances the expression of IFN-β and interferon-stimulated genes (ISGs) in infected primary macrophages . These findings suggest that p17 could be a potential target for developing attenuated ASFV vaccine candidates.
The data from siRNA experiments showed:
| Gene | Expression change with p17 knockdown |
|---|---|
| IFN-β | Significant upregulation (p < 0.01) |
| ISG15 | Significant upregulation (p < 0.01) |
| ISG56 | Significant upregulation (p < 0.01) |
| IL-6 | Significant upregulation (p < 0.05) |
| IL-8 | Significant upregulation (p < 0.05) |
| Viral p72 | Significant downregulation (p < 0.01) |
Researchers exploring p17 manipulation for vaccine development should consider:
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Gene deletion approaches to create p17-deficient or modified ASFV strains
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Balancing attenuation with sufficient immunogenicity
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Evaluating safety through monitoring for potential reversion to virulence
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Assessing protective efficacy against challenge with virulent ASFV strains
How does p17 interact with other ASFV proteins during viral assembly and maturation?
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Proximity labeling techniques to identify p17's protein interaction network
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Cryo-electron microscopy to visualize p17's position within the virion structure
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Time-course analyses to determine when and where p17 is incorporated during virion assembly
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Conditional expression systems to assess virion formation in the absence of functional p17
What are the optimal cell models for studying p17 function?
For different experimental objectives, researchers should consider:
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Primary PAMs: Best for studying authentic host-pathogen interactions and validating key findings
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Stable cell lines: Useful for high-throughput screening and preliminary mechanistic studies
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Heterologous expression systems: Appropriate for isolated protein-protein interaction studies
When designing experiments with these cellular models, researchers should carefully control for cell type-specific differences in STING expression levels and IFN signaling pathways.
How should researchers approach p17 gene manipulation for functional studies?
Several complementary approaches can be used to manipulate p17 expression and function:
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RNA interference: siRNAs targeting the D117L gene have proven effective for transient knockdown studies . When using this approach, researchers should design at least 2-3 different siRNAs to control for off-target effects and validate knockdown efficiency at both mRNA and protein levels.
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Inducible expression systems: Placing the D117L gene under control of an inducible promoter allows for temporal control of expression . The synthetic promoter derived from the late p72 promoter has been successfully used for this purpose.
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Domain mutants: Creating specific mutations in the transmembrane domain (amino acids 39-59) can help dissect structure-function relationships .
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Recombinant virus generation: For studying p17 in the context of viral infection, researchers should consider generating recombinant ASFV with modifications to the D117L gene.
What are the technical challenges in detecting p17-STING interactions?
Investigating p17-STING interactions presents several technical challenges that researchers should address:
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Low abundance detection: p17 is not readily detectable in total cell extracts and requires immunoprecipitation for reliable detection .
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Membrane protein solubilization: Both p17 and STING are membrane proteins, requiring careful optimization of detergent conditions for extraction while preserving native interactions.
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Timing considerations: STING undergoes dynamic changes in localization and modification upon activation, so interaction studies should consider appropriate time points post-stimulation.
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Confirmation with multiple methods: Co-immunoprecipitation results should be validated with complementary approaches such as proximity ligation assays or fluorescence resonance energy transfer (FRET).
How should researchers interpret apparent contradictions in p17 functional studies?
When analyzing p17 function, researchers may encounter seemingly contradictory results due to:
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Strain differences: Functional variations in p17 between different ASFV isolates
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Cell type effects: Different cellular backgrounds affecting p17-host protein interactions
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Assay sensitivity: Variations in detection methods for protein interactions or signaling outcomes
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Timing considerations: Different time points of analysis affecting observed phenotypes
To address these challenges, researchers should:
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Clearly specify the ASFV strain used in all experiments
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Include appropriate positive and negative controls
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Use multiple complementary assays to confirm key findings
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Conduct time-course experiments to capture dynamic processes
What statistical approaches are most appropriate for analyzing p17 knockdown effects?
When analyzing the effects of p17 knockdown on viral replication and host gene expression, researchers should:
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Perform experiments with at least three biological replicates to account for variability
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Use appropriate statistical tests (e.g., Student's t-test for pairwise comparisons, ANOVA for multiple comparisons)
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Apply multiple testing corrections when analyzing effects on numerous host genes
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Report both statistical significance (p-values) and effect sizes to provide a complete picture of biological relevance
For time-course data, consider using repeated measures ANOVA or mixed-effects models rather than multiple t-tests to maintain appropriate statistical power.
How might p17 function be targeted for novel ASFV therapeutics?
The identification of p17 as an immune evasion protein suggests several potential therapeutic strategies:
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Small molecule inhibitors: Compounds that disrupt the p17-STING interaction without affecting normal STING function could potentially restore antiviral immunity during ASFV infection.
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Peptide-based approaches: Designing peptides that mimic the STING binding region to competitively inhibit p17 binding.
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RNA therapeutics: siRNA or antisense oligonucleotides targeting D117L expression could be explored as potential antiviral strategies.
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Structure-based drug design: As structural information about p17 becomes available, rational drug design approaches could be employed.
Researchers exploring these approaches should establish robust screening assays that specifically measure the p17-STING interaction or its downstream effects on IFN signaling.
What are the knowledge gaps regarding p17's role in ASFV pathogenesis in vivo?
Despite advances in understanding p17's molecular functions, several knowledge gaps remain regarding its role in ASFV pathogenesis in vivo:
Addressing these questions will require:
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Development of recombinant ASFV strains with modified p17
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Controlled in vivo infection studies using these recombinants
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Comprehensive immune profiling in infected animals
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Comparative genomics across ASFV strains with different virulence profiles
