Recombinant African swine fever virus Envelope protein p54

What is the ASFV p54 protein and what gene encodes it?

P54 is an African swine fever virus inner-membrane protein encoded by the E183L gene. It serves critical functions in viral internalization, transport, and assembly processes. The protein is structurally significant for ASFV particles and participates in key interactions with host cellular machinery, making it an important target for vaccine development and antiviral strategies . Functionally, p54 plays a pivotal role in the binding of viral particles to target cells and subsequent infection processes, with antibodies against p54 shown to significantly weaken ASFV's ability to infect cells .

How does p54 interact with host cellular components during infection?

P54 directly binds to the 8 kDa light-chain cytoplasmic dynein (LC8), a component of the microtubule motor complex. This interaction is critical for the virus's ability to hijack the cellular transport machinery during infection . The binding occurs both in vitro and within infected cells, with p54 and LC8 colocalizing specifically at the microtubular organizing center during viral infection . This interaction represents a molecular mechanism by which ASFV exploits the host's microtubule-mediated transport system to facilitate viral movement within the cytoplasm, which is essential for effective viral replication and assembly .

What specific domain of p54 is responsible for its interaction with dynein?

A remarkably compact 13-amino-acid domain within p54 is sufficient for binding to the LC8 light chain of cytoplasmic dynein. Within this domain, an SQT motif has been identified as critical for this binding interaction . This highly specific interaction domain highlights the precise molecular mechanisms that viruses have evolved to exploit host cellular machinery. The identification of this binding domain provides valuable information for researchers developing targeted interventions that might disrupt this virus-host interaction .

What methods are most effective for producing recombinant p54 for immunological studies?

For successful production of recombinant p54, researchers have effectively utilized expression systems in HEK-293T cells with subsequent purification for ELISA analyses . The methodology typically includes:

• Gene cloning and insertion into appropriate expression vectors

• Addition of purification tags (such as 6xHis-tag) at both N- and C-termini for effective isolation

• Transfection into mammalian expression systems

• Protein purification via affinity chromatography

• Verification of protein integrity through Western blotting and functionality assays

In the cited research, the purified p54 protein served as a substrate for ELISA analysis, enabling precise detection of p54-specific antibodies generated during immunization studies . This methodological approach ensures high-quality antigenic material for downstream immunological investigations.

How can researchers effectively engineer viral vectors expressing functional p54?

Engineering viral vectors expressing functional p54 has been successfully accomplished using CRISPR/Cas9 technology combined with homologous recombination. A detailed methodology includes:

• Designing expression constructs with appropriate promoters (e.g., EF1α promoter has been effective)

• Adding purification tags (6xHis-tag) to both N- and C-termini of p54

• Co-transfecting plasmid (5 μg) and homologous recombinant fragment (1 μg) into suitable cells (e.g., 293T cells)

• Adding the base virus (e.g., 5×10^5 TCID50 of PRV-ΔTK/ΔgE) 6 hours post-transfection

• Collecting viral culture when cytopathic effects are observed in approximately 90% of cells

• Amplifying and purifying the recombinant virus through multiple rounds of phagocytosis

• Confirming genetic stability through multiple passages (at least 40 generations)

• Verifying p54 gene presence and integrity via PCR and Sanger sequencing using specific primers

This approach has been demonstrated to produce stable recombinant viruses that effectively express p54 while maintaining desired characteristics of the vector virus .

How does p54 contribute to protective immunity against ASFV?

P54 is a significant immunogen that stimulates protective antibody responses against ASFV. When expressed in recombinant viral vectors such as pseudorabies virus (PRV), p54 induces robust antigen-specific IgG production as early as 7 days post-vaccination . The immunological significance of p54 stems from its role in viral internalization and assembly, making antibodies against this protein particularly effective at neutralizing viral infectivity .

Studies have demonstrated that vaccination with recombinant vectors expressing p54 (such as PRV-∆TK-(CD2v)-∆gE-(p54)) successfully induces p54-specific antibodies via intramuscular vaccination, which may confer protection against ASFV infection . This immune response helps establish immunological memory that can potentially prevent or reduce the severity of ASFV infection, highlighting p54's value in vaccine development strategies.

What is the optimal combination of ASFV antigens for recombinant vaccine development?

Current research indicates that combining p54 with other ASFV antigens, particularly CD2v, produces more robust immune responses than using p54 alone. The recombinant PRV-∆TK-(CD2v)-∆gE-(p54) virus, which expresses both ASFV CD2v and p54 proteins, has demonstrated promising results as a potential bivalent vaccine candidate .

This combination approach offers several advantages:

• It targets multiple viral components simultaneously

• It induces stronger antibody responses against both antigens

• The distinct functions of each protein (CD2v's role in hemadsorption and p54's role in viral transport) provide complementary protective mechanisms

• The dual-antigen approach helps overcome potential variations in individual immune responses

The synergistic effect of combining these antigens represents a more comprehensive approach to vaccine development against ASFV, potentially providing broader and more effective protection .

How does the p54-LC8 interaction mechanistically facilitate ASFV replication?

The interaction between p54 and the light chain of cytoplasmic dynein (LC8) represents a sophisticated molecular hijacking of host cellular transport mechanisms. This interaction occurs through a specific binding domain containing an essential SQT motif within p54 . Mechanistically, this binding allows ASFV to exploit the minus-end-directed microtubule-associated motor protein function of dynein, facilitating targeted transport of viral components through the cytoplasm .

Research using p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, demonstrated impeded ASFV infection, providing strong evidence for dynein's essential role in the viral lifecycle . The p54-LC8 interaction facilitates transport of viral components to the microtubular organizing center, where the two proteins have been shown to colocalize during infection . This strategic localization likely promotes viral assembly and subsequent steps in the replication cycle.

This molecular mechanism demonstrates how ASFV has evolved specific protein interactions to exploit existing cellular machinery, providing insights that could be targeted for antiviral intervention strategies.

What structural modifications to recombinant p54 might enhance its immunogenicity while maintaining functional epitopes?

Enhancing p54 immunogenicity while preserving functional epitopes requires careful structural considerations. Based on current research, several strategic approaches can be considered:

• Tag placement optimization: Studies have successfully utilized Flag-tag and 6xHis-tag at both N- and C-termini of p54 for detection and purification without compromising immunogenicity . Future research could explore alternative tag configurations that might further enhance immune recognition.

• Promoter selection: The EF1α promoter has proven effective for driving p54 expression in recombinant viral vectors . Comparative studies with alternative promoters could identify optimal expression levels that maximize immunogenicity.

• Epitope preservation: Special attention must be paid to preserving the 13-amino-acid domain containing the SQT motif that interacts with LC8, as this region represents a functionally significant epitope that may be important for generating neutralizing antibodies .

• Expression system selection: While HEK-293T cells have been successfully used for p54 expression and purification , exploring alternative expression systems might yield proteins with different post-translational modifications that could influence immunogenicity.

These considerations should guide researchers in designing recombinant p54 constructs that balance optimal immune stimulation with preservation of key functional domains.

How might genetic variation in p54 across ASFV strains impact recombinant vaccine efficacy?

Genetic variation in p54 across different ASFV strains poses significant challenges for developing broadly protective recombinant vaccines. Although the search results don't directly address strain variations, this question merits careful consideration in vaccine development strategies. Researchers should:

• Conduct comprehensive sequence analyses across geographically diverse ASFV isolates to identify conserved regions within p54 that could serve as universal vaccine targets

• Evaluate whether critical functional domains, such as the 13-amino-acid LC8-binding region containing the SQT motif , are conserved across strains

• Consider developing polyvalent vaccines incorporating p54 variants from predominant circulating strains if significant variation exists

• Employ experimental challenge studies using heterologous ASFV strains to assess cross-protection efficacy of recombinant p54-based vaccines

• Investigate whether combining p54 with other ASFV antigens, such as CD2v , might broaden protection against diverse strains by targeting multiple viral components simultaneously

This strategic approach to addressing genetic variation would strengthen the potential for developing recombinant p54-based vaccines with broad efficacy against multiple ASFV strains.

What are the optimal conditions for detecting p54-specific antibodies in serological assays?

Developing sensitive and specific serological assays for p54-specific antibodies requires careful optimization. Based on successful research approaches, the following methodology is recommended:

• Antigen preparation: Express and purify p54 from mammalian cells (such as HEK-293T) to ensure proper folding and post-translational modifications . The addition of 6xHis-tags facilitates efficient purification while maintaining antigenic properties.

• ELISA protocol optimization:

  • Coating concentration: 1-5 μg/ml of purified p54 protein

  • Blocking solution: 5% non-fat milk or BSA in PBS-T

  • Sample dilution: Serial dilutions starting from 1:100 for serum samples

  • Detection system: Anti-species IgG conjugated with HRP

  • Substrate development: TMB with optimization of development time

• Cross-reactivity considerations: Include appropriate controls to distinguish p54-specific antibodies from antibodies against other ASFV proteins or related viral antigens

• Timing of detection: Significant serum IgG responses against p54 can be detected as early as 7 days post-vaccination with recombinant vectors expressing p54 , with continued monitoring recommended through day 14 and beyond

These methodological approaches have successfully demonstrated the immunogenicity of p54 in recombinant vector systems and provide a framework for reliable detection of p54-specific antibodies in research and diagnostic applications .

What strategies can overcome challenges in expressing functional recombinant p54 in bacterial systems?

While the search results primarily discuss mammalian expression systems for p54, bacterial expression systems often present challenges for viral membrane proteins. Researchers working with p54 in bacterial systems should consider these strategies:

• Codon optimization: Adapt the p54 coding sequence to preferred codon usage in the bacterial host to enhance expression efficiency

• Fusion partners: Utilize solubility-enhancing fusion partners (such as MBP, GST, or SUMO) to improve protein folding and reduce inclusion body formation

• Expression conditions optimization:

  • Reduced temperature (16-25°C) during induction

  • Lower IPTG concentrations (0.1-0.5 mM)

  • Rich media formulations with osmotic stabilizers

  • Co-expression with chaperone proteins

• Refolding protocols: If inclusion bodies form, develop optimized solubilization and refolding protocols using gradual removal of denaturants coupled with oxidative refolding conditions

• Functional domain focus: Consider expressing only the functionally critical domains of p54, such as the 13-amino-acid LC8-binding region , rather than the full-length protein

• Membrane mimetics: Include detergents or lipid nanodiscs during purification to stabilize the membrane-associated regions of p54

These approaches can help overcome the typical challenges encountered when expressing viral membrane proteins in bacterial systems, potentially enabling more cost-effective production of p54 for research and vaccine development.

How might CRISPR/Cas9 technology be further optimized for engineering more effective p54-expressing recombinant viral vectors?

CRISPR/Cas9 technology has already proven valuable for engineering recombinant viral vectors expressing p54 . Future optimizations could include:

• Improved guide RNA design: Utilizing machine learning algorithms to design guide RNAs with minimal off-target effects while maximizing on-target efficiency for precise insertion of p54 expression cassettes

• Alternative Cas variants: Exploring Cas variants with higher fidelity (such as HiFi Cas9 or Cas12a) to reduce off-target modifications in viral vectors

• Multiplexed editing: Simultaneous modification of multiple loci within viral vectors to create optimized expression platforms for p54 along with other ASFV antigens

• Inducible or tissue-specific promoters: Engineering more sophisticated expression control systems for p54 to enhance safety profiles and immunogenicity of recombinant vectors

• Homology-directed repair templates: Designing improved homologous recombination templates with longer homology arms and optimized p54 expression cassettes to increase integration efficiency

• Selection marker strategies: Developing more efficient selection systems for identifying successfully modified viral vectors expressing p54

These advanced CRISPR/Cas9 approaches would build upon the successful methodologies already demonstrated , potentially yielding next-generation viral vectors with enhanced safety, stability, and efficacy for p54-based vaccine development.

What potential exists for developing p54-based diagnostic tools for early ASFV detection?

The unique properties of p54 provide promising opportunities for developing sensitive and specific diagnostic tools for early ASFV detection. Future research directions might include:

• Lateral flow assays: Developing field-deployable immunochromatographic tests using anti-p54 antibodies for rapid detection of ASFV p54 in clinical samples

• Aptamer-based biosensors: Selecting and optimizing DNA or RNA aptamers that specifically bind p54 for incorporation into electrochemical or optical biosensors

• PCR-based detection: Designing optimized primers targeting the p54-encoding E183L gene for use in qPCR or isothermal amplification assays

• Multiplex detection platforms: Integrating p54 detection with other ASFV markers (such as CD2v) to enhance diagnostic sensitivity and specificity

• CRISPR-based diagnostics: Adapting CRISPR-Cas systems (such as Cas12a or Cas13) for specific detection of E183L gene sequences with smartphone-compatible readout systems

• Serological platforms: Developing improved ELISA or microsphere immunoassay formats using recombinant p54 to detect antibody responses indicating recent or ongoing ASFV infection

These diagnostic approaches would leverage the extensive knowledge of p54 structure and function to create tools that could substantially improve ASFV surveillance and control efforts, complementing the vaccine development approaches described earlier.