Recombinant African swine fever virus Transmembrane protein EP84R (Pret-066)

What is African swine fever virus Transmembrane protein EP84R (Pret-066)?

African swine fever virus (ASFV) Transmembrane protein EP84R (Pret-066) is an essential viral protein embedded in the inner envelope that surrounds the viral core. The protein functions as a crucial molecular bridge linking the internal genome-containing core to the external viral layers . This small transmembrane polypeptide (83 amino acids) is encoded by the EP84R gene and plays a vital role in viral morphogenesis by targeting core shell polyproteins to the inner viral envelope, enabling subsequent genome packaging and nucleoid formation .

How is recombinant pEP84R typically produced and stored for experimental use?

Recombinant full-length pEP84R is commonly expressed in E. coli with an N-terminal His-tag to facilitate purification . The optimized production process yields >90% purity as determined by SDS-PAGE analysis . For laboratory storage and handling:

What is the subcellular localization of pEP84R during viral infection?

Immunofluorescence and immunoelectron microscopy studies reveal that pEP84R displays distinct localization patterns:

• In viral factories (assembly sites within infected cells)

• In ER-derived precursor viral membranes

• At the interface between viral core shell and inner envelope in immature and mature particles

• In punctate structures throughout the cytoplasm (likely dispersed virus particles)

• In mature budding particles at the plasma membrane

When expressed independently of other viral proteins, pEP84R predominantly localizes to the nuclear envelope, Golgi complex, and ER membrane cisternae, as confirmed by double-labeling with ER markers such as PDI .

What specific role does pEP84R play in ASFV morphogenesis?

pEP84R functions as a critical molecular link in ASFV assembly:

• Core Shell Assembly Guidance: pEP84R targets core shell polyproteins (pp220 and pp62) to the inner viral envelope

• Genome Packaging Facilitation: The correct targeting of core shell proteins by pEP84R enables DNA packaging and nucleoid formation

• Structural Integrity Maintenance: Absence of pEP84R results in non-infectious core-less icosahedral particles with significant DNA-packaging defects

• Proteolytic Processing Regulation: pEP84R is required for proper proteolytic processing of pp220 and pp62 polyproteins

Studies using a recombinant ASFV with inducible EP84R expression (vEP84Ri) demonstrated that without pEP84R, over 90% of viral particles are defective in core shell and nucleoid organization, confirming its essential role in viral assembly .

How does pEP84R interact with viral polyproteins to facilitate virion assembly?

pEP84R specifically interacts with core shell polyproteins through several mechanisms:

• Direct Binding: Co-immunoprecipitation assays demonstrate that pEP84R binds to the N-terminal region of polyprotein pp220

• Subcellular Targeting: When co-expressed with pp220, both proteins colocalize to perinuclear areas and the cell surface, while pp220 alone accumulates in discrete cytoplasmic areas

• Complex Formation: Co-expression of pEP84R, pp220, and pp62 results in significant colocalization at perinuclear areas and discrete plasma membrane patches

• Membrane Guidance: pEP84R directs pp220 and pp62 to ER-derived membranes, which is essential for core shell assembly

This interaction is functionally critical, as the absence of pEP84R during viral infection leads to severe impairment of pp220 and pp62 proteolytic processing and mislocalization of these proteins to non-ER membranes .

What experimental approaches are most effective for studying pEP84R function?

Researchers have successfully employed multiple complementary approaches to elucidate pEP84R function:

These approaches collectively provide a comprehensive toolkit for investigating the structural and functional aspects of pEP84R in ASFV biology.

What phenotype results from pEP84R deficiency during viral replication?

The absence of pEP84R during ASFV infection produces a distinctive phenotype with profound defects in virion assembly:

• Structural Abnormalities:

  • Formation of non-infectious core-less icosahedral particles

  • Significant DNA-packaging defects

  • Normal outer capsid and outer envelope formation

  • Disorganized particulate material in the core region

  • Some particles contain acentric dense nucleoid surrounded by asymmetric core shell

• Aberrant Core Shell Structures:

  • Accumulation of core shell-like "zipper structures"

  • Attachment to cytosolic side of plasma membrane

  • Association with endosomal and lysosomal compartments

  • Formation of parallel stacked arrays within viral factories

Quantitative analysis shows that under non-permissive conditions, >90% of vEP84Ri particles are defective in core shell and nucleoid organization, compared to <10% defective particles under permissive conditions .

How might pEP84R serve as a target for antiviral development?

pEP84R represents a promising target for therapeutic development against ASFV for several reasons:

• Essential Function: pEP84R is indispensable for producing infectious virus particles

• Critical Interactions: The specific interaction between pEP84R and core shell polyproteins represents a virus-specific process that could be disrupted

• Membrane Localization: As a transmembrane protein at the interface between viral components, pEP84R may have domains accessible to small molecule inhibitors

• Unique Role: Its function in targeting core shell polyproteins to the inner viral envelope is critical for nucleoid formation and genome packaging

These characteristics position pEP84R as a key target for potential therapeutic interventions against this devastating disease that currently affects domestic and wild pigs across Africa, Europe, Asia, Oceania, and the Americas .

What molecular techniques are recommended for analyzing pEP84R interactions with other viral components?

For comprehensive analysis of pEP84R interactions with viral components, researchers should consider these methodological approaches:

• Protein-Protein Interaction Analysis:

  • Co-immunoprecipitation with epitope-tagged pEP84R (e.g., Flag-tagged)

  • Truncation mutants to map minimal interaction domains

  • Site-directed mutagenesis to identify critical residues

• Imaging-Based Interaction Studies:

  • Multi-color immunofluorescence with antibodies to pEP84R and potential partners

  • High-resolution microscopy to precisely map spatial relationships

  • Live-cell imaging with fluorescently-tagged proteins

• Biochemical Characterization:

  • Membrane fractionation to determine co-fractionation patterns

  • Triton X-114 phase separation to distinguish membrane association properties

  • Protease protection assays to map topology of interactions

• Functional Validation:

  • Inducible expression systems (e.g., IPTG-controlled)

  • Trans-complementation assays

  • Competitive inhibition approaches

These techniques can be combined to build a comprehensive understanding of how pEP84R orchestrates the assembly of viral components during ASFV morphogenesis.

Comparison of ASFV Particles Under Different pEP84R Expression Conditions

Recombinant pEP84R Protein Specifications