Recombinant African swine fever virus Uncharacterized protein D129L (Ba71V-103)

What is the D129L protein of African swine fever virus?

The D129L protein (pD129L) is a nonstructural protein of African swine fever virus with 129 amino acids. It functions as an immune evasion factor, specifically antagonizing type I interferon (IFN) responses and IFN-stimulated gene (ISG) expression. In the HLJ/2018 strain of ASFV, the protein is encoded by the D129L gene. The protein plays a crucial role in helping ASFV evade host antiviral immunity, which contributes to the virus's pathogenicity .

How is D129L expressed during viral infection?

Expression analysis demonstrates that D129L is a late-transcribed gene of ASFV. Its transcription profile closely resembles that of B646L (encoding p72), which is another late-expressed gene, but differs significantly from early-transcribed genes like CP204L (encoding p30). This temporal regulation can be experimentally verified using cytosine arabinoside (Arac), an inhibitor of DNA replication and gene transcription. When porcine alveolar macrophages (PAMs) are infected with ASFV in the presence of Arac, the expression of pD129L is significantly inhibited at 24 hours post-infection, while early proteins like p30 remain unaffected .

What is the significance of the Ba71V strain designation for D129L research?

The Ba71V strain has played a foundational role in ASFV research as it was the first ASFV genome to be completely sequenced. This avirulent strain was derived through adaptation of the highly virulent BA71 strain to grow in Vero cells. The genome of Ba71V, compared to its virulent parent strain, contains specific mutations that contribute to its attenuated phenotype. The D129L protein from the Ba71V strain (Ba71V-103) serves as an important reference for understanding how genetic variations in this protein might contribute to virulence differences among ASFV isolates .

What molecular mechanism does D129L use to antagonize interferon responses?

The D129L protein employs a sophisticated mechanism to antagonize type I interferon production. Specifically, pD129L:

• Localizes to the nucleus of infected cells

• Binds directly to transcriptional coactivators CBP/p300

• Inhibits the interaction between interferon regulatory factor 3 (IRF3) and CBP/p300

• Suppresses activation of the IFN-β promoter, thereby reducing IFN-β induction

Importantly, pD129L does not affect the expression of adaptor proteins in the cGAS-STING signaling pathway or the nuclear translocation of IRF3, but rather specifically targets the final step of IRF3-mediated transcriptional activation .

Which domains of pD129L are critical for its immunosuppressive function?

Structure-function analysis has identified the HrcA domain of pD129L as crucial for its interaction with host proteins. This domain specifically binds to the IRF3-binding domain of p300, thereby competitively inhibiting the formation of the IRF3-p300 complex that is essential for type I interferon gene expression. This mapping of functional domains provides potential targets for the development of antiviral strategies that could specifically neutralize the immunosuppressive activity of pD129L without affecting other viral functions .

How can the immunomodulatory function of D129L be experimentally verified?

To experimentally verify the immunomodulatory function of D129L, researchers have employed several complementary approaches:

• RNA interference: Knockdown of D129L by specific small interfering RNAs enhances IFN-β mRNA transcription in ASFV-infected primary porcine alveolar macrophages (PAMs) .

• Reporter assays: Cotransfection of HEK293T cells with plasmids expressing pD129L, along with IFN-β promoter-luciferase reporter constructs and cGAS-STING expression plasmids, demonstrates that pD129L significantly antagonizes IFN-β promoter activity .

• Protein-protein interaction studies: Co-immunoprecipitation and immunofluorescence microscopy confirm that pD129L specifically interacts with CBP/p300 in the nucleus and disrupts the IRF3-p300 interaction .

What expression systems are effective for producing recombinant D129L protein?

While multiple expression systems can be used for recombinant ASFV protein production, viral vector-based systems have shown particular promise. The Semliki Forest virus (SFV) vector system has been successfully employed for expressing ASFV antigens, including structural proteins p32 and p54. This system involves:

• Construction of a replicon plasmid containing the D129L gene

• In vitro transcription of replicon RNA

• Electroporation of RNA into baby hamster kidney (BHK-21) cells along with helper RNA

• Collection of replication-defective viral particles expressing the target protein

This approach could be adapted for D129L expression, particularly for immunological studies or vaccine development .

What purification strategies are most effective for recombinant D129L protein?

Based on protocols used for other ASFV proteins, an effective purification strategy for recombinant D129L would include:

• Lysis of cells under native or denaturing conditions depending on downstream applications

• Initial purification using affinity chromatography (if the recombinant protein includes a tag such as His6)

• Additional purification steps such as ion exchange chromatography or size exclusion chromatography to remove contaminants

• Validation of protein purity by SDS-PAGE and western blotting using anti-D129L antibodies or anti-tag antibodies

• Functional validation through binding assays with known interaction partners (e.g., p300)

Optimization of these conditions would be necessary for each specific expression system .

How might D129L be utilized in ASFV diagnostic assays?

While D129L has not been widely used in diagnostic assays to date, its potential as a diagnostic target should be considered in light of its consistent expression across ASFV isolates. Other ASFV proteins, including E183L/p54, K205R, A104R/histone-like, and B602L, have demonstrated 100% sensitivity as serological diagnostic antigens by 21 days post-infection . Based on this precedent, recombinant D129L could be evaluated for:

• Development of ELISA-based serological assays to detect anti-D129L antibodies in infected animals

• Potential inclusion in multiplex diagnostic platforms that simultaneously detect antibodies against multiple ASFV proteins

• Use in competitive ELISA formats to distinguish infected from vaccinated animals (DIVA assays)

The late expression profile of D129L may make it particularly valuable for detecting persistent or chronic infections .

What is the potential of D129L as a target for vaccine development?

Given D129L's role in immune evasion, targeting this protein could be a promising approach for ASFV vaccine development:

• Subunit vaccines: Recombinant D129L, potentially combined with other immunogenic ASFV proteins, could be used in subunit vaccine formulations.

• Live-attenuated vaccines: Deletion or mutation of the D129L gene could contribute to viral attenuation while potentially enhancing immune responses through increased type I interferon production.

• Vector-based vaccines: Expression of D129L in viral vectors, similar to the SFV system used for p32 and p54, could induce antibody and T-cell responses against this protein.

How can gene editing approaches be used to study D129L function?

CRISPR/Cas9 and related gene editing technologies offer powerful tools to investigate D129L function:

• Knockout studies: Generation of D129L-deleted ASFV mutants to determine the impact on viral replication and pathogenesis in vitro and in vivo.

• Domain mapping: Introduction of targeted mutations in the HrcA domain or other functional regions to precisely define structure-function relationships.

• Reporter knock-in: Introduction of fluorescent tags at the endogenous D129L locus to track protein expression and localization during infection without overexpression artifacts.

• Host factor modification: Editing of host factors like p300 to introduce mutations in the D129L-binding region to confirm interaction specificity and functional relevance.

These approaches would provide more definitive evidence for D129L function than the current knockdown and overexpression studies .

What comparative genomic approaches reveal insights about D129L conservation and evolution?

The BA71 and BA71V genomes provide a valuable model for understanding ASFV evolution and attenuation. Comparative analysis reveals that while these strains have the smallest genomes for virulent and attenuated ASFV respectively, they are essentially identical except for a relatively small number of changes. Analysis of D129L conservation across diverse ASFV isolates could reveal:

• Selection pressures on different domains of the protein

• Correlation between D129L sequence variations and viral virulence

• Potential host adaptation signatures in the D129L sequence

• Identification of invariant regions that might be critical for function and thus good targets for intervention

This evolutionary perspective would complement functional studies and guide rational design of antivirals or vaccines targeting D129L .

What are the main challenges in studying D129L protein interactions and how can they be overcome?

Studying protein-protein interactions involving D129L presents several technical challenges:

Implementation of these methodological approaches would help overcome the limitations of current studies and provide a more comprehensive understanding of D129L's interaction network .

How can structural biology approaches enhance our understanding of D129L function?

Despite the significant functional characterization of D129L, its three-dimensional structure remains unknown. Several structural biology approaches could provide valuable insights:

• X-ray crystallography: Determination of the crystal structure of D129L alone and in complex with p300 fragments would reveal the precise molecular basis for this interaction.

• Cryo-electron microscopy: Analysis of larger complexes involving D129L, particularly those that may be difficult to crystallize.

• NMR spectroscopy: Characterization of dynamic regions and potential conformational changes upon binding to interaction partners.

• Computational structural prediction: Application of AlphaFold or similar algorithms to predict the structure of D129L and validate through experimental approaches.

Structural information would facilitate structure-based drug design targeting the D129L-p300 interaction and provide a framework for understanding how sequence variations in different ASFV isolates might affect function .

How might combined immunomodulatory strategies targeting D129L improve ASFV vaccine development?

Given D129L's role in suppressing interferon responses, several innovative approaches could be explored:

• Development of ASFV vaccine candidates with modified D129L to reduce immunosuppression while maintaining immunogenicity

• Combination of D129L-targeted interventions with other immunomodulatory strategies addressing different immune evasion mechanisms of ASFV

• Evaluation of whether neutralizing D129L function could serve as an adjuvant strategy for existing ASFV vaccine candidates

• Investigation of whether antibodies against D129L contribute to protective immunity or primarily serve as diagnostic markers

These approaches would need to be carefully balanced, as complete elimination of immunomodulatory functions might reduce viral replication too severely for live-attenuated vaccine approaches .

What is the potential interplay between D129L and other ASFV immunomodulatory proteins?

ASFV encodes multiple proteins that modulate host immune responses, suggesting potential functional redundancy or synergy. Future research should investigate:

• Potential interactions between D129L and other ASFV immunomodulatory proteins such as E120R

• Whether D129L functions independently or as part of larger viral protein complexes

• The temporal regulation of different immune evasion strategies during the ASFV replication cycle

• Whether targeting multiple immunomodulatory proteins simultaneously could overcome viral immune evasion more effectively than targeting individual factors

Understanding these interactions would provide a more comprehensive picture of ASFV pathogenesis and potentially reveal more effective intervention strategies .