Recombinant African swine fever virus Envelope protein p54 (Ba71V-126)

What is the African Swine Fever Virus p54 protein and what is its role in viral structure?

The p54 protein is a 25-kDa structural polypeptide encoded by the E183L gene of ASFV, essential for viral morphogenesis. Despite its name, p54 has a molecular weight of approximately 25 kDa, with the designation relating to its relative position in two-dimensional gels . It functions as a type I membrane-anchored protein containing a transmembrane domain near its N-terminus and forms disulfide-linked homodimers through its unique luminal cysteine .

The protein localizes to the endoplasmic reticulum (ER)-derived envelope precursors and is crucial for recruiting and transforming ER membranes into viral envelope precursors . Experiments using a lethal conditional recombinant virus (vE183Li) demonstrated that suppression of p54 synthesis arrests virus morphogenesis at an extremely early stage, before membrane precursor formation, resulting in discrete electron-lucent areas essentially free of viral structures at virus factories .

What are the main functional domains of p54, and how do they contribute to virus-host interactions?

The p54 protein contains several critical functional domains with distinct roles in virus-host interactions:

The transmembrane domain enables p54 targeting to ER membranes when expressed in transfected cells, while the DLC8 binding domain interacts with the LC8 subunit of cytoplasmic dynein, facilitating microtubule-mediated virus transport . The identified B-cell epitopes are highly conserved among ASFV strains and induce humoral immune responses, making them valuable targets for vaccine development .

How does p54 interact with the host cell's microtubular motor complex, and what are the implications for viral trafficking?

The p54 protein establishes a high-affinity interaction with the dynein light chain (DYNLL1/DLC8) of the microtubular motor complex through a specific binding domain . This interaction represents a sophisticated mechanism for microtubule-mediated virus transport that facilitates the movement of viral components to viral factories near the microtubule organizing center (MTOC) .

Research has revealed that the p54-dynein interaction is strong enough to form a stable molecular weight complex in vitro . The binding interface was mapped using nuclear magnetic resonance (NMR) spectroscopy, showing specific residues on DLC8 that directly interact with p54 . This interaction is functionally significant, as demonstrated by experiments with synthetic peptides mimicking the p54-dynein binding domain (DBD) that compete with and disrupt this interaction .

When cells are treated with these inhibitory peptides, there is a measurable decrease in viral infectivity, replication, and production . These findings provide strong evidence that p54-mediated interactions with the host cell's microtubular transport system are critical for successful ASFV infection, offering potential targets for antiviral intervention.

What is the mechanism of p54's involvement in virus envelope formation, and how does it recruit ER membranes?

The p54 protein plays a pivotal role in the recruitment and transformation of endoplasmic reticulum (ER) membranes into viral envelope precursors through a complex mechanism . During ASFV assembly at cytoplasmic virus factories, p54 initiates the formation of precursor membranous structures derived from collapsed cisternal domains of the surrounding ER .

The process involves several coordinated steps:

• ER targeting: p54 contains a transmembrane domain that effectively targets it to ER membranes when expressed in transfected cells .

• Membrane modification: Once localized to the ER, p54 facilitates the recruitment and transformation of these membranes into specialized structures that serve as precursors for the viral envelope .

• Structural organization: p54 forms disulfide-linked homodimers through its unique luminal cysteine, which contributes to the structural organization of the membrane precursors .

Experimental evidence using the conditional lethal recombinant vE183Li demonstrates that when p54 synthesis is repressed, virus morphogenesis is arrested before the formation of precursor membranes . The virus factories appear as discrete electron-lucent areas essentially devoid of viral structures, while aberrant zipper-like structures formed by unprocessed core polyproteins accumulate in close association with ER cisternae outside the assembly sites . These observations confirm that p54 is critical for the initial stages of viral envelope formation.

What are the most effective protocols for expressing and purifying recombinant p54 protein for structural and functional studies?

Effective expression and purification of recombinant p54 involves several carefully optimized protocols to ensure protein functionality and stability:

Expression System Selection:
The bacterial expression system using BL21(DE3) E. coli transformed with a p54-encoding plasmid (such as pET-p54ΔTM) has proven effective . For proper expression, the transmembrane domain is typically removed (p54ΔTM) to enhance solubility while maintaining functional domains .

Expression Protocol:

• Transform BL21(DE3) cells with the p54ΔTM-containing plasmid

• Grow transformed cells in LB medium until reaching optimal density

• Induce protein expression with 1 mM IPTG for 3 hours at 37°C

• Harvest cells by centrifugation at 4,000 × g for 20 minutes at 4°C

Purification Strategy:

• Resuspend cell pellet in lysis buffer (typically PBS with protease inhibitors)

• Disrupt cells by sonication and lysozyme treatment (1 mg/ml)

• Clear cell debris by ultracentrifugation

• Purify His-tagged p54 using metal affinity chromatography (TALON metal affinity agarose)

• Elute purified protein with 200 mM imidazole

• Dialyze three times against PBS overnight at 4°C to remove imidazole

For structural studies requiring labeled protein, minimal media supplemented with 15N-labeled ammonium chloride can be used for NMR spectroscopy applications .

Quality Control Measures:

• SDS-PAGE analysis to verify purity and molecular weight

• Western blotting with anti-p54 antibodies for identity confirmation

• Functional assays to confirm binding to known partners (e.g., DLC8 binding assay)

This methodology has been successfully employed in studies investigating p54-DLC8 interactions and for identifying epitopes through structural analysis .

What techniques are most suitable for analyzing p54 interactions with host cell components during infection?

Several sophisticated techniques have proven effective for studying p54 interactions with host cell components:

1. Nuclear Magnetic Resonance (NMR) Spectroscopy:
NMR has been successfully employed to map the binding interface between p54 and dynein light chain (DLC8). Using 15N-labeled DLC8 and unlabeled p54, researchers tracked changes in chemical shifts to identify residues involved in binding. The protocol involves:

• Recording spectra on a 600 MHz spectrometer with a cryogenic probe

• Using sweep widths of 12 ppm (1H) and 30 ppm (15N)

• Collecting data points (128 indirect and 2,048 direct) with 48 scans

• Processing with specialized software like TOPSPIN

2. Co-immunoprecipitation and Pull-down Assays:
These techniques allow identification of protein-protein interactions:

• Use purified p54 as bait with cellular lysates

• Capture complexes with anti-p54 antibodies or through affinity tags

• Identify interacting partners by mass spectrometry or western blotting

3. Proximity Ligation Assays (PLA):
This method detects protein interactions in situ with high sensitivity:

• Fix infected cells at various time points

• Use primary antibodies against p54 and suspected interacting proteins

• Apply PLA probes and perform ligation and amplification

• Visualize interaction sites by fluorescence microscopy

4. Live-cell Imaging with Fluorescently Tagged Proteins:
Monitoring dynamics of p54 during infection:

• Generate recombinant ASFV expressing p54-GFP fusion protein

• Track movement in relation to cellular structures (ER, microtubules)

• Perform time-lapse microscopy to follow trafficking patterns

5. Competitive Peptide Inhibition:
This approach has been particularly valuable for validating specific interaction domains:

• Design peptides mimicking the DLC8 binding domain of p54

• Introduce these peptides into cells prior to infection

• Measure changes in viral infectivity, replication, and trafficking

• Use control peptides with irrelevant sequences as experimental controls

The combination of these techniques has revealed critical interactions, such as the high-affinity binding between p54 and DLC8, which mediates intracellular transport of the virus .

How can identified p54 epitopes be utilized for the development of subunit vaccines against ASFV?

The identified p54 epitopes provide valuable targets for ASFV subunit vaccine development through several strategic approaches:

Epitope-Based Vaccine Design:
Several key epitopes identified on p54, including TMSAIENLR and 76QQWVEV81 , have demonstrated strong immunogenicity. These epitopes are highly conserved across ASFV strains, making them excellent candidates for broad-spectrum vaccine development. Researchers can utilize these findings by:

• Incorporating multiple epitopes into a single construct to enhance immune response breadth

• Designing peptide vaccines featuring these specific sequences

• Creating chimeric proteins that present these epitopes in optimal conformations

Recombinant Vector Systems:
Viral vectors expressing p54 have shown promise as potential vaccine platforms. For example, research has demonstrated successful expression of p54 (E183L) in recombinant pseudorabies virus (PRV) . This approach offers several advantages:

• The ASFV p54 protein can be stably inherited and expressed in the recombinant virus after multiple passages

• Viral vectors can efficiently deliver the antigen to antigen-presenting cells

• This system can elicit both humoral and cell-mediated immune responses

Multi-Antigen Approaches:
Studies indicate that combining p54 with other ASFV immunogenic proteins enhances protective efficacy:

Delivery System Optimization:
The method of epitope delivery significantly impacts vaccine efficacy. Several systems have been tested for p54-based vaccines:

• Semliki Forest Virus replicon particles (SFV-RPs) expressing p54 (SFV-p54) have demonstrated the ability to elicit high IL-4 expression in mice, indicating a strong Th1-type response

• Recombinant PRV vectors expressing p54 along with other ASFV antigens have shown stable inheritance and protein expression through multiple passages

These approaches demonstrate that p54 epitopes, particularly when strategically combined with other ASFV antigens and delivered via appropriate vector systems, hold significant promise for subunit vaccine development against ASFV.

What are the most sensitive and specific methods for detecting p54 antibodies in diagnostic applications?

Several methodologies have been optimized for detecting p54 antibodies with high sensitivity and specificity in diagnostic applications:

Enzyme-Linked Immunosorbent Assay (ELISA):
Custom p54-specific ELISAs have been developed with excellent diagnostic performance:

• Microplates coated with recombinant p54 antigen capture anti-p54 antibodies from serum samples

• Detection systems typically employ anti-ASF monoclonal antibody-biotin conjugates and HRP-tagged streptavidin

• After substrate addition and color development, absorbance is measured at 450 nm

• Sensitivity can reach 96-98% in field conditions with optimized protocols

Multiplex Bead-Based Immunoassays:
These assays allow simultaneous detection of antibodies against multiple ASFV proteins:

• p54 protein is coupled to microspheres with distinct spectral signatures

• Reactions with serum antibodies are detected using fluorescently labeled secondary antibodies

• Flow cytometry analysis provides quantitative results

• This method offers improved throughput compared to traditional ELISAs

Lateral Flow Assays (LFAs):
Rapid point-of-care tests have been developed based on p54:

• Utilize colloidal gold-conjugated p54 protein or anti-p54 antibodies

• Provide results within 10-15 minutes without specialized equipment

• Particularly valuable for field diagnostics in resource-limited settings

• Though slightly less sensitive than laboratory ELISAs, they offer crucial rapid detection capabilities

Western Blotting for Confirmatory Testing:
While not ideal for high-throughput screening, western blotting provides highly specific confirmation:

• Recombinant p54 or viral lysates are separated by SDS-PAGE

• After transfer to membranes, detection with test sera and labeled secondary antibodies

• P54-specific bands at approximately 25 kDa confirm positive results

• This approach is particularly valuable for resolving ambiguous ELISA results

Diagnostic relevance of p54 is enhanced by its early expression during infection and its high immunogenicity. Combined with tests for other ASFV proteins like p30 and p72, p54-based diagnostics form the cornerstone of comprehensive ASFV serological detection systems .

What are the current contradictions or knowledge gaps in understanding p54 function during ASFV infection?

Despite significant advances in p54 research, several important contradictions and knowledge gaps remain:

Contradictory Findings on Cytoskeletal Requirements:
Conflicting results have been reported regarding the role of the actin cytoskeleton in ASFV entry and infection:

• Some studies report that disruption of the actin cytoskeleton with cytochalasin D significantly alters infectivity

• Contradictory findings suggest minimal effects on infectivity when using other actin-disrupting agents like jasplakinolide and latrunculin A

• These discrepancies may result from differences in experimental conditions, viral strains, or cell types, highlighting the need for standardized approaches

Incomplete Understanding of p54 Post-Translational Modifications:
While p54 is known to form disulfide-linked homodimers, other potential modifications remain poorly characterized:

• The complete spectrum of post-translational modifications affecting p54 function is unknown

• How these modifications might be regulated during different stages of infection remains to be elucidated

• Whether host cell factors can modulate these modifications as a defense mechanism is unclear

Knowledge Gaps in p54 Interactions with Host Immune System:
Several aspects of how p54 interfaces with host immunity remain unexplored:

• The complete repertoire of host receptors recognizing p54 epitopes is not fully characterized

• How p54-specific antibodies contribute to viral neutralization versus potential enhancement is incompletely understood

• The relative immunodominance of different p54 epitopes across diverse host genetic backgrounds requires further investigation

Mechanistic Details of p54 in Membrane Recruitment:
While p54 is essential for recruiting ER membranes to form viral envelope precursors, the precise molecular mechanisms remain unclear:

• The specific ER proteins or lipids targeted by p54 remain unidentified

• How p54 induces membrane curvature or other structural changes is not well understood

• The coordination between p54 and other viral factors in this process needs further characterization

Addressing these knowledge gaps will require innovative experimental approaches combining structural biology, advanced imaging techniques, and systems biology to build a more comprehensive model of p54 function in ASFV infection.

What emerging technologies and methodological approaches might advance our understanding of p54 in ASFV pathogenesis?

Several cutting-edge technologies and methodological approaches show promise for advancing p54 research:

Cryo-Electron Microscopy (Cryo-EM) and Tomography:
These techniques can revolutionize our understanding of p54's structural role:

• Visualize p54 in its native conformation within viral particles

• Map the precise localization of p54 during different stages of virion assembly

• Determine structural changes induced by p54 in host membranes during envelope formation

• Resolution at near-atomic level can reveal critical protein-protein interactions

CRISPR-Cas9 Genome Editing for Host Factor Identification:
This approach can systematically identify host factors interacting with p54:

• Create genome-wide knockout libraries in susceptible cell lines

• Screen for altered ASFV replication or p54 localization

• Validate hits through complementation studies and biochemical approaches

• Identify potential therapeutic targets among critical host factors

Proximity Labeling Proteomics:
These methods can map the dynamic p54 interactome during infection:

• Express p54 fused to enzymes like BioID or APEX2 that biotinylate proximal proteins

• Apply at different stages of infection to capture temporal changes in interactions

• Identify previously unknown binding partners through mass spectrometry

• Validate key interactions using orthogonal techniques

Single-Cell Technologies:
These approaches can reveal cell-to-cell variation in p54 function:

• Single-cell RNA-seq to identify host transcriptional responses to p54

• Mass cytometry (CyTOF) to measure multiple cellular parameters simultaneously

• Microfluidic devices to isolate and analyze individual infected cells

• Correlate p54 expression levels with cellular outcomes

Advanced Imaging Techniques:
Next-generation imaging can provide unprecedented insights:

• Super-resolution microscopy to visualize p54 distribution below the diffraction limit

• Live-cell lattice light-sheet microscopy to track p54 dynamics in real-time

• Correlative light and electron microscopy (CLEM) to link functional observations with ultrastructural details

• Expansion microscopy to physically enlarge specimens for enhanced resolution

Structural Vaccinology Approaches:
Rational design based on p54 structure can advance vaccine development:

• Computational epitope prediction and optimization

• Structure-based design of conformationally stable p54 immunogens

• Nanoparticle display of multiple p54 epitopes in defined orientations

• Synthetic biology approaches to create completely artificial immunogens based on p54 epitopes

The integration of these advanced technologies promises to resolve current contradictions and fill knowledge gaps in our understanding of p54 biology, potentially leading to breakthrough interventions against ASFV.

What are the most promising avenues for translating p54 research into practical applications for ASFV control?

The most promising translational avenues for p54 research include:

• Subunit and epitope-based vaccines leveraging the highly conserved and immunogenic regions of p54, particularly when combined with other ASFV antigens in multivalent formulations.

• Peptide inhibitors targeting the p54-dynein interaction, which have demonstrated the ability to reduce viral infectivity by disrupting intracellular transport mechanisms.

• Improved diagnostic tools based on p54 epitopes, enabling more sensitive and specific detection of ASFV infection in field conditions.

• Viral vector platforms expressing p54 alongside other ASFV antigens, which have shown promise in eliciting protective immune responses.

• Structure-based drug design targeting critical p54 functions in viral assembly and host interaction.