Recombinant African swine fever virus Uncharacterized membrane protein KP93L (Pret-001)

What is known about the basic structure of ASFV Pret-001 protein?

Pret-001 (KP93L) is an uncharacterized membrane protein from African swine fever virus isolated from Tick/South Africa/Pretoriuskop Pr4/1996. While the complete crystal structure has not been reported in current literature, we know it belongs to a family of ASFV membrane proteins . Unlike some well-characterized ASFV proteins such as pS273R (which has a catalytic triad C232-H168-N187 and consists of both core and arm domains), the detailed structural information for Pret-001 remains limited .

Methodological approach for structural characterization:

• Express recombinant protein using cell-free expression systems

• Conduct circular dichroism (CD) spectroscopy to determine secondary structure elements

• Utilize X-ray crystallography or cryo-EM techniques for tertiary structure determination

• Apply computational structure prediction using deep learning approaches as demonstrated with other membrane proteins

What experimental systems are appropriate for studying Pret-001 function?

The primary experimental systems for studying ASFV proteins include:

When studying Pret-001, researchers should consider that ASFV naturally targets macrophages. Porcine alveolar macrophages represent the gold standard for studying ASFV protein function in a native cellular environment, though they present challenges for large-scale experimentation .

How should researchers design experiments to assess Pret-001 localization within infected cells?

When investigating protein localization of an uncharacterized ASFV membrane protein like Pret-001, researchers should implement a systematic approach similar to that used for other ASFV proteins such as p10:

• Generate fluorescently tagged constructs (GFP/mCherry-Pret-001 fusion proteins)

• Perform time-course experiments in both infected and transfected cells

• Use subcellular fractionation combined with Western blotting to confirm microscopy findings

• Compare localization patterns at different stages of viral infection (early vs. late)

For comprehensive analysis, compare results with known ASFV proteins. For example, the p10 protein of ASFV strongly accumulates in the nucleus during late stages post-infection and exhibits DNA-binding activities related to its helix-turn-helix structural motif .

Example experimental timeline:

• 24 hours post-transfection or infection: Early localization assessment

• 48-72 hours post-transfection or infection: Late-stage localization assessment

• Use counterstaining with markers for cellular compartments (nucleus, ER, Golgi, plasma membrane)

What controls are essential when studying Pret-001 expression and function?

Designing rigorous controls is critical for studying uncharacterized proteins like Pret-001:

When implementing an siRNA approach to study protein function, include a non-targeting siRNA control (siNC) as demonstrated in ASFV studies focusing on gasdermin proteins . Additionally, when assessing cell viability and death, employ multiple complementary assays (e.g., WST-1 for viability and LDH release for cell death) .

How can researchers determine if Pret-001 forms functional multimers or complexes with other viral or host proteins?

Investigating protein-protein interactions for membrane proteins like Pret-001 requires multiple complementary approaches:

• Co-immunoprecipitation studies: Express tagged versions of Pret-001 in relevant cell types and identify binding partners through mass spectrometry analysis.

• Proximity labeling approaches: Utilize BioID or APEX2 fusion constructs to identify proteins in close proximity to Pret-001 during infection.

• Cross-linking mass spectrometry: Apply chemical cross-linkers to stabilize transient interactions before protein complex isolation.

• Split reporter systems: Fuse Pret-001 and candidate interacting proteins with complementary fragments of reporters like luciferase or fluorescent proteins.

For example, to identify caspase interactions with viral proteins, researchers have utilized co-expression systems where "Flag-GSDMA or HA-caspase-3 alone, or both were coexpressed in HEK293T cells, and the interaction and subcellular colocalization of these two proteins were examined" . Similar methodologies could be applied to study Pret-001 interactions.

What approaches should be used to determine if soluble analogues of Pret-001 can be developed for structural studies?

Membrane proteins present significant challenges for structural studies. Recent advances in computational design offer promising approaches for creating soluble functional analogues:

• Deep learning pipeline application: Apply robust deep learning algorithms to design soluble versions of membrane proteins while maintaining critical structural features .

• Domain-focused approach: Identify specific functional domains that might be expressed independently while maintaining native folding.

• Verification methodology:

  • Biophysical analyses to confirm thermal stability of designed soluble analogues

  • Experimental structure determination to verify design accuracy

  • Functional assays to determine if soluble versions retain key activities

Recent research has demonstrated "high thermal stability of the designs and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, standing as a proof-of-concept for bringing membrane protein functions to the soluble proteome" . This approach could potentially be applied to create soluble versions of Pret-001 for easier structural characterization.

How does Pret-001 potentially contribute to ASFV virulence and host immune evasion?

While specific functions of Pret-001 are not well characterized in the provided literature, researchers can design experiments based on approaches used for other ASFV proteins:

• Gene knockout/knockdown studies: Generate recombinant viruses lacking Pret-001 or use siRNA approaches to assess impact on viral replication and host response.

• Host pathway analysis: Investigate effects on key immune signaling pathways, similar to how some ASFV proteins like pS273R have been shown to potentially "antagonize the host IFN-I pathway by deubiquitinating specific proteins" .

• Comparative virulence studies: Compare virulence between wild-type and Pret-001 mutant strains in appropriate models, measuring parameters such as:

  • Fever progression (e.g., increased rectal temperature from day 3 post-challenge)

  • Survival rates at different viral doses

  • Viral genome copies in tissues

  • Inflammatory marker expression

For example, ASFV BA71 isolate studies demonstrated that "in vivo inoculation of BA71 was highly pathogenic, causing severe ASF clinical signs, including high fever from day 3 post-challenge to the end of the experiment and independently of the dose used" . Similar comparative studies could reveal Pret-001's role in virulence.

What methodologies are appropriate for investigating Pret-001's potential role in viral entry or membrane fusion?

For membrane proteins potentially involved in viral entry:

• Entry inhibition assays: Generate antibodies or peptides targeting Pret-001 and assess their ability to block viral entry.

• Liposome fusion assays: Reconstitute purified Pret-001 into liposomes and measure membrane fusion events under varying conditions.

• Time-of-addition experiments: Add Pret-001-targeting reagents at different timepoints during infection to determine when the protein functions.

• Super-resolution microscopy: Track fluorescently labeled Pret-001 during early infection events to visualize its localization during entry.

• Site-directed mutagenesis: Create point mutations in predicted functional domains to identify regions essential for viral entry.

What is the potential of Pret-001 as a subunit vaccine candidate against African swine fever?

Evaluating Pret-001 as a vaccine candidate requires systematic assessment:

• Immunogenicity profiling:

  • Measure antibody responses to recombinant Pret-001 in immunized animals

  • Characterize T-cell responses to predict cell-mediated immunity

  • Evaluate cross-reactivity against multiple ASFV strains

• Protective efficacy studies:

  • Design challenge studies with appropriate controls

  • Measure parameters including viral load, clinical signs, and survival rates

  • Compare with other subunit vaccine candidates

• Combination approaches:

  • Test Pret-001 in combination with other ASFV antigens

  • Evaluate various adjuvant formulations to enhance immune responses

The challenges faced with live attenuated virus (LAV) approaches highlight the need for alternative strategies. While LAVs have been tested, "given the failure of LAVs, additional tools are yet needed" , suggesting subunit vaccines targeting well-conserved proteins like Pret-001 could be valuable alternatives.

How can researchers optimize expression systems for producing recombinant Pret-001 for vaccine studies?

The optimization of expression systems is critical for obtaining sufficient quantities of properly folded Pret-001:

For Pret-001 specifically, cell-free expression systems have been successfully employed , though the protein's membrane-associated nature presents challenges. When optimizing expression, researchers should:

• Test multiple expression constructs with varying tags and fusion partners

• Carefully evaluate protein solubility and proper folding

• Implement quality control steps including size exclusion chromatography and functional assays

How might researchers investigate the role of Pret-001 in modulating host cell death pathways during ASFV infection?

Recent research has revealed complex interactions between ASFV and host cell death pathways, particularly pyroptosis. To investigate Pret-001's potential role:

• Knockdown/knockout studies:

  • Use siRNA targeting Pret-001 during ASFV infection

  • Measure impact on cell death markers using LDH release assays

  • Assess inflammatory cytokine release (e.g., IL-1β) by ELISA

• Protein interaction studies:

  • Investigate potential interactions with cell death pathway components

  • Test for interactions with gasdermin proteins, which are cleaved during ASFV infection

  • Examine possible interplay with caspase activation pathways

• Time-course analysis:

  • Compare cell death kinetics between wild-type and Pret-001-deficient conditions

  • Determine if Pret-001 affects early or late stages of cell death

Studies have shown that "ASFV infection induced GSDMA expression in porcine alveolar macrophages (PAMs). Subsequently, GSDMA was cleaved by caspase-4" . Similar methodologies could be applied to determine if Pret-001 influences these pathways.

What bioinformatic approaches can help predict functional domains within the largely uncharacterized Pret-001?

For uncharacterized proteins like Pret-001, computational approaches can provide valuable insights:

• Sequence-based analysis:

  • Multiple sequence alignment across ASFV isolates to identify conserved regions

  • Identification of transmembrane domains and signal sequences

  • Prediction of post-translational modifications

• Structure prediction:

  • Apply deep learning approaches such as AlphaFold2 to predict tertiary structure

  • Identify potential functional domains by structural comparison

  • Model membrane integration and topology

• Functional annotation:

  • Search for distant homologs with known functions

  • Predict binding sites for DNA, RNA, or other proteins

  • Identify potential catalytic sites or structural motifs

• Evolutionary analysis:

  • Assess evolutionary conservation across ASFV strains

  • Identify regions under selective pressure

  • Compare with homologs in related viruses

Similar approaches have revealed important insights about other ASFV proteins, such as p10, which contains "a characteristic helix-turn-helix structural motif" and has "the C-terminal helix rich in lysine residues and the serine-rich residues found in the N-terminal helix... crucial for the interaction with dsDNA" .