Recombinant African swine fever virus Transmembrane protein EP84R (War-064)

A: The most effective genetic approach for studying pEP84R function involves creating conditional lethal mutants using inducible expression systems. As demonstrated in current research, a recombinant virus named vEP84Ri has been developed where pEP84R expression is controlled by IPTG (isopropyl β-D-1-thiogalactopyranoside) . This experimental system places the EP84R gene under the control of an IPTG-inducible promoter, creating a conditional lethal mutant that only produces infectious virus in the presence of the inducer.

The methodology involves:

• Engineering the recombinant virus with the inducible system

• Infecting cells (such as Vero cells or porcine macrophages) with the recombinant virus

• Comparing virus growth in permissive (with IPTG) versus non-permissive (without IPTG) conditions

• Analyzing viral phenotypes through:

  • Plaque formation assays

  • One-step growth curves

  • Western blot to verify protein expression

  • Electron microscopy to observe virion structure

  • qPCR to quantify viral DNA content in particles

This approach has revealed that in the absence of pEP84R expression, the virus fails to produce infectious particles, confirming the protein's essential nature for viral viability .

A: Electron microscopy (EM) is the gold standard for investigating pEP84R's role in ASFV morphogenesis. EM analysis of cells infected with conditional pEP84R mutants (vEP84Ri) under both permissive and non-permissive conditions reveals critical structural differences in viral particles .

Under non-permissive conditions (when pEP84R is not expressed), EM reveals the presence of large quantities of defective particles with the following characteristics:

• Core-less icosahedral structures

• Properly formed outer capsid layers

• Absence of the nucleoid-containing core

• Accumulation in assembly sites and budding areas

In contrast, under permissive conditions, EM shows complete virions with:

• Well-defined core shells

• Properly formed nucleoids containing the viral genome

• Complete multilayered architecture

The stark differences observable through EM provide direct visual evidence of pEP84R's essential role in core assembly. This technique should be conducted at approximately 18 hours post-infection for optimal visualization of the assembly defects . For even more detailed analysis, immunoelectron microscopy using gold-labeled antibodies against pEP84R could potentially track the protein's precise localization during virion assembly.

A: Quantitative PCR (qPCR) provides the most direct measurement of the impact of pEP84R deficiency on viral genome packaging. This technique has demonstrated that virions produced under pEP84R-deficient conditions contain approximately four times less viral DNA than those produced when pEP84R is properly expressed .

The experimental approach involves:

• Purifying viral particles from cells infected with vEP84Ri under both permissive (pEP84R+) and non-permissive (pEP84R-) conditions

• Extracting DNA from equivalent amounts of viral particles

• Performing qPCR with primers targeting specific ASFV genomic regions

• Normalizing results to account for particle numbers

• Comparing relative DNA content between pEP84R+ and pEP84R- particles

Complementary biochemical approaches could include:

• Western blot analysis to examine the incorporation of core proteins in purified virions

• Immunoprecipitation studies to identify protein-protein interactions with pEP84R

• Density gradient centrifugation to separate and characterize different particle populations

Together, these approaches establish that pEP84R plays a crucial role in the genome packaging process during ASFV assembly .

A: Investigating interactions between pEP84R and other ASFV structural proteins requires a multi-faceted approach:

• Co-immunoprecipitation (Co-IP) assays:

  • Express recombinant pEP84R with a fusion tag (e.g., His-tag)

  • Use anti-pEP84R antibodies or anti-tag antibodies for pull-down experiments from infected cell lysates

  • Identify co-precipitated proteins through mass spectrometry

  • Confirm specific interactions with targeted Western blots

• Proximity-based labeling techniques:

  • Generate fusion constructs of pEP84R with BioID or APEX2

  • Express these constructs in infected cells during ASFV assembly

  • Identify proximal proteins through streptavidin pull-down and mass spectrometry

• Fluorescence resonance energy transfer (FRET):

  • Create fluorescently-tagged versions of pEP84R and candidate interacting partners

  • Analyze energy transfer between fluorophores in live or fixed cells during infection

  • Quantify interaction strength through FRET efficiency calculations

• Structural biology approaches:

  • Crystallize pEP84R alone or in complex with binding partners

  • Use cryo-electron microscopy to visualize pEP84R in the context of the viral particle

  • Employ cross-linking mass spectrometry to map interaction interfaces

These techniques would help identify pEP84R's interaction partners within the viral assembly complex, particularly proteins involved in core shell formation and nucleoid packaging, which could explain its essential role in viral morphogenesis .

A: Given pEP84R's essential role in ASFV morphogenesis, particularly its function in core assembly and genome packaging , it represents a promising target for anti-ASFV therapeutics. Research strategies to explore this potential include:

• High-throughput screening approaches:

  • Develop assays using recombinant pEP84R to screen compound libraries

  • Design screening assays that can detect:

    • Direct binding to pEP84R

    • Inhibition of pEP84R interactions with other viral proteins

    • Interference with pEP84R membrane insertion or localization

• Structure-based drug design:

  • Determine the three-dimensional structure of pEP84R through X-ray crystallography or NMR

  • Identify potential binding pockets or interaction interfaces

  • Use in silico screening to identify compounds that could bind to these sites

  • Validate hits through biochemical and cellular assays

• Peptide-based inhibitors:

  • Design peptides that mimic interaction interfaces of pEP84R

  • Test their ability to compete with natural interactions

  • Optimize lead peptides for stability and cellular uptake

• Genetic validation experiments:

  • Use the established vEP84Ri conditional system to validate compound effects

  • Compare phenotypes of chemical inhibition with genetic repression

  • Establish dose-response relationships for promising compounds

• Development of resistance assays:

  • Generate ASFV variants under selective pressure from lead compounds

  • Sequence resistant viruses to identify escape mutations

  • Use this information to refine inhibitor design

A combination of these approaches could lead to the identification of molecules that specifically disrupt pEP84R function, potentially resulting in non-infectious virus particles similar to those observed under genetic repression conditions .

A: Purifying functional recombinant pEP84R presents several challenges due to its transmembrane nature. The following table outlines major challenges and recommended solutions:

The most successful approach documented uses E. coli expression systems with N-terminal His-tags (10xHis), followed by purification in Tris-based buffers supplemented with glycerol or trehalose . For applications requiring native conformation, consider reconstitution into nanodiscs or proteoliposomes after purification to better mimic the natural membrane environment.

A: Accurately assessing pEP84R mutations requires a comprehensive experimental pipeline that integrates genetic, biochemical, and microscopic approaches:

• Generation of mutant constructs:

  • Design targeted mutations based on sequence conservation, predicted structural features, or known functional domains

  • Create point mutations, deletions, or chimeric constructs using site-directed mutagenesis

  • Incorporate mutations into the inducible expression system (similar to vEP84Ri)

• Complementation assays:

  • Infect cells with the conditional pEP84R mutant virus (vEP84Ri) under non-permissive conditions

  • Provide mutant pEP84R variants through trans-expression

  • Quantify the ability of each variant to rescue viral production

• Quantitative assessment metrics:

  • Viral titer determination through plaque assays or TCID50

  • One-step growth curves to measure replication kinetics

  • qPCR quantification of viral DNA content in particles

  • Western blot analysis of viral protein incorporation into particles

• Structural characterization:

  • Electron microscopy to visualize particle morphology defects

  • Immuno-EM to localize mutant proteins within viral structures

  • Super-resolution microscopy to track assembly dynamics

• Biochemical interaction analysis:

  • Co-immunoprecipitation with known binding partners

  • Membrane integration assays to assess transmembrane domain function

  • Thermal shift assays to evaluate protein stability

This systematic approach provides multiple readouts of protein function, allowing researchers to distinguish between mutations affecting protein stability, localization, protein-protein interactions, or specific assembly steps. The combination of quantitative (viral titers, qPCR) and qualitative (EM, microscopy) data provides a comprehensive view of how specific amino acid residues contribute to pEP84R's essential role in ASFV morphogenesis .

A: While pEP84R is specific to African swine fever virus, comparative analysis with functional analogs in other large DNA viruses can provide evolutionary insights:

• Sequence and structural comparison:

  • pEP84R is an 83-amino acid transmembrane protein with specific topology

  • Comparative sequence analysis with other viral transmembrane proteins that function in particle assembly can reveal:

    • Conserved motifs that may indicate functional importance

    • Structural similarities despite sequence divergence

    • Patterns of selection pressure on different protein domains

• Functional comparison with other viral assembly factors:

  • pEP84R's role in connecting the viral core to outer layers is conceptually similar to:

    • Poxvirus A16 protein, which links core and membrane during morphogenesis

    • Herpesvirus UL25, which connects capsid to tegument proteins

  • These functional analogs, despite lacking sequence homology, may represent convergent evolution toward solving similar assembly challenges

• Evolutionary analysis across ASFV isolates:

  • Compare EP84R sequences from different ASFV isolates to identify:

    • Degree of conservation across viral strains

    • Isolate-specific variations and their potential functional implications

    • Correlation between sequence variations and virulence differences

• Host adaptation signatures:

  • Examine whether EP84R sequences show adaptations based on host species

  • Compare sequences from viruses isolated from domestic pigs versus natural reservoir hosts (warthogs, bushpigs)

  • Identify potential signatures of host adaptation that might influence assembly efficiency

This comparative analysis would provide insights into the evolution of viral assembly mechanisms and potentially identify conserved features that could serve as broader targets for antiviral development against large DNA viruses.

A: Systems biology offers powerful approaches to contextualize pEP84R within the broader landscape of ASFV infection:

• Interactome mapping:

  • Use high-throughput protein-protein interaction screens to place pEP84R in the viral protein interaction network

  • Identify both viral and host cell proteins that interact with pEP84R

  • Construct interaction networks to visualize pEP84R's position within assembly complexes

• Temporal-spatial proteomics:

  • Apply pulse-labeling techniques to track the dynamics of pEP84R expression and localization

  • Use proximity labeling to identify proteins co-localizing with pEP84R at different infection stages

  • Develop a temporal map of assembly factor recruitment coordinated with pEP84R expression

• Multi-omics integration:

  • Combine data from:

    • Proteomics of pEP84R interactions

    • Transcriptomics of genes up/downregulated when pEP84R is repressed

    • Metabolomics to identify changes in cellular metabolism

  • Develop computational models of assembly pathways incorporating pEP84R function

• Cryo-electron tomography studies:

  • Visualize the three-dimensional architecture of assembling virions with and without pEP84R

  • Map the precise location of pEP84R within the inner envelope

  • Understand how pEP84R facilitates core formation at the molecular level

• Single-particle tracking:

  • Use fluorescently tagged pEP84R to track its movement in living infected cells

  • Correlate its dynamics with the progression of viral assembly

  • Identify recruitment patterns and temporal association with other viral components

These systems approaches would provide a comprehensive understanding of how pEP84R functions within the complex, multi-step process of ASFV assembly, potentially revealing additional roles beyond its established function in core formation and genome packaging .

A: The discovery that pEP84R deficiency leads to the production of non-infectious virus-like particles with intact outer structure but defective cores presents a promising avenue for vaccine development:

• Advantages of pEP84R-deficient particles as vaccine candidates:

  • Particles maintain outer structural proteins that can elicit protective immune responses

  • Genomic DNA content is significantly reduced (approximately 4-fold less) , enhancing safety

  • Core-less particles are inherently non-infectious but preserve antigenic epitopes

  • The outer capsid structure remains intact, potentially preserving important conformational epitopes

• Production strategies:

  • Use the inducible vEP84Ri system under non-permissive conditions to generate defective particles

  • Develop stable cell lines expressing all essential structural proteins except pEP84R

  • Engineer production systems with precisely controlled levels of pEP84R to tune particle properties

• Immunogenicity assessment protocol:

  • Compare immune responses to:

    • Wild-type virions (inactivated)

    • pEP84R-deficient particles

    • Individual viral proteins

  • Evaluate both humoral and cell-mediated immune responses

  • Test protective efficacy against challenge with virulent ASFV strains

• Adjuvant and delivery optimization:

  • Test various adjuvant formulations to enhance immunogenicity

  • Explore different delivery routes (intramuscular, intradermal, mucosal)

  • Develop prime-boost strategies combining pEP84R-deficient particles with other approaches

• Safety and efficacy validation:

  • Confirm complete absence of replication-competent virus

  • Establish minimum protective dose

  • Determine duration of immunity

  • Assess cross-protection against heterologous ASFV strains

This approach could overcome some of the challenges associated with current ASFV vaccine development efforts by providing particles that closely mimic the structure of infectious virions while eliminating the risk of infection, thereby addressing both safety and efficacy concerns .