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

What is pEP84R and what is its structural composition?

pEP84R is a transmembrane polypeptide embedded in the inner envelope that surrounds the viral core of African swine fever virus. The protein consists of 83 amino acids with the sequence: "MPYSRDITKFITATEPEVGLPLLALQRSKSVIGIILLVISLLLIFIGIIILSVSSHTTAG SVLVVLSLILGGGGFFLIYKDNS" . As a transmembrane protein, it has hydrophobic regions that traverse the membrane and hydrophilic segments that interact with the aqueous environment. The structural analysis reveals that pEP84R contains multiple transmembrane domains that allow it to anchor within the endoplasmic reticulum (ER)-derived inner envelope of the virus .

What is the role of pEP84R in ASFV morphogenesis?

pEP84R plays a crucial role in ASFV core assembly by targeting the core shell polyproteins to the inner viral envelope, which enables subsequent genome packaging and nucleoid formation . This protein acts as a molecular guide that ensures proper localization of vital structural components during viral assembly. Research has demonstrated that pEP84R specifically binds to the N-terminal region of the polyprotein pp220, directing it and pp62 to the ER membranes where viral assembly occurs . This targeting mechanism is essential for the formation of the viral core shell, which serves as a bridging layer required for the formation of the central genome-containing nucleoid .

How does pEP84R differ from other ASFV structural proteins?

Unlike other structural proteins that primarily serve as building blocks for the viral capsid or nucleocapsid, pEP84R functions as a regulatory protein that coordinates the assembly process. While capsid proteins like p72 are directly involved in forming the icosahedral outer structure, pEP84R serves as an essential mediator that directs the assembly of core components at the correct cellular location . Furthermore, whereas many structural proteins are incorporated into the mature virion as integral components, pEP84R's primary role appears to be in the assembly process, specifically in the targeting and organization of polyproteins pp220 and pp62 to the inner envelope .

How can researchers express and purify recombinant pEP84R for laboratory studies?

For laboratory studies, recombinant pEP84R can be expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification . The methodology involves:

• Cloning the EP84R gene (1-83aa) into an appropriate expression vector

• Transforming E. coli with the recombinant vector

• Inducing protein expression (typically with IPTG)

• Lysing cells and purifying the protein using nickel affinity chromatography

• Further purification through size exclusion chromatography if needed

• Concentration and storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0

For reconstitution, it is recommended to centrifuge the vial briefly before opening and reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% is advised for long-term storage at -20°C/-80°C to avoid repeated freeze-thaw cycles that may compromise protein integrity .

What techniques are most effective for studying pEP84R interactions with other viral proteins?

Based on research methodologies employed in the field, the following techniques have proven effective for studying pEP84R interactions:

• Co-immunoprecipitation assays: These have successfully demonstrated that pEP84R binds to the N-terminal region of pp220 . The procedure involves:

  • Expressing tagged versions of pEP84R and potential interacting proteins

  • Preparing cell lysates under non-denaturing conditions

  • Using antibodies against the tag to pull down protein complexes

  • Analyzing precipitated proteins by Western blotting

• Confocal microscopy with fluorescently-tagged proteins: This technique allows visualization of the co-localization of pEP84R with other viral proteins in infected cells or co-transfection experiments .

• Yeast two-hybrid screening: Can be used to identify novel protein-protein interactions.

• Recombinant virus systems: The generation of conditional mutant viruses (like vEP84Ri) where pEP84R expression can be controlled has been instrumental in understanding its function .

How can researchers analyze the impact of pEP84R absence on viral assembly?

To analyze the impact of pEP84R absence on viral assembly, researchers can employ a combination of techniques as demonstrated in the literature:

• Generation of inducible expression systems: Creating recombinant viruses where pEP84R expression can be controlled, such as the vEP84Ri derived from the BA71V strain .

• Electron microscopy: To visualize morphological changes in viral particles when pEP84R is absent, revealing the formation of core-less icosahedral particles .

• DNA packaging assays: To quantify the DNA-packaging defect associated with pEP84R absence .

• Infectivity assays: To measure the reduction in viral infectivity when pEP84R is not expressed .

• Immunofluorescence microscopy: To track the mistargeting of core shell proteins to non-ER membranes in the absence of pEP84R .

Table 1: Comparison of Viral Particles Formed With and Without pEP84R Expression

What molecular mechanisms allow pEP84R to target core shell polyproteins to the inner viral envelope?

The molecular mechanisms by which pEP84R targets core shell polyproteins to the inner viral envelope involve specific protein-protein interactions and membrane localization properties:

• Direct binding to polyproteins: Co-immunoprecipitation assays have shown that pEP84R specifically binds to the N-terminal region of pp220, one of the major core shell polyproteins . This direct interaction serves as the primary mechanism for recruiting these polyproteins to the assembly site.

• ER membrane integration: As a transmembrane protein, pEP84R integrates into the ER membrane, which becomes the inner viral envelope. Its transmembrane domains anchor it firmly in the membrane, creating a bridge between the membrane and core components .

• Scaffolding function: pEP84R likely serves as a molecular scaffold that correctly positions pp220 and pp62 polyproteins at the inner envelope, enabling their proper co-assembly into core shell structures .

• Sequential assembly guidance: Evidence suggests that pEP84R facilitates a sequential assembly process where core shell formation precedes and is required for proper genome packaging and nucleoid formation .

When pEP84R is absent, these mechanisms fail, resulting in mistargeting of core shell polyproteins and formation of aberrant structures at incorrect cellular locations .

How does the absence of pEP84R affect DNA packaging in ASFV particles?

The absence of pEP84R has profound effects on DNA packaging in ASFV particles through a cascade of assembly defects:

• Disruption of core shell formation: Without pEP84R, the core shell polyproteins pp220 and pp62 are mistargeted to non-ER membranes, preventing proper core shell assembly .

• Loss of genome packaging scaffold: The core shell normally serves as a scaffold for genome packaging. Its improper formation in the absence of pEP84R eliminates the structural framework required for DNA incorporation .

• Formation of empty particles: Research shows that without pEP84R, ASFV forms non-infectious, core-less icosahedral particles with a significant DNA-packaging defect . These particles maintain their outer capsid structure but lack the internal components necessary for genome containment.

• Disruption of nucleoid formation: The nucleoid, which contains the viral genome, fails to form properly without the correctly assembled core shell, further compromising DNA packaging .

This evidence indicates that pEP84R indirectly affects DNA packaging by ensuring the proper formation and localization of the core shell components that are necessary for subsequent genome incorporation .

What is the relationship between pEP84R and the viral polyproteins pp220 and pp62?

The relationship between pEP84R and the viral polyproteins pp220 and pp62 reveals a coordinated assembly mechanism:

• Direct binding interaction: pEP84R directly binds to the N-terminal region of pp220 as demonstrated by co-immunoprecipitation assays . This physical interaction is essential for proper targeting of pp220.

• Co-assembly direction: When pEP84R is co-expressed with both pp220 and pp62, it leads to the formation of ER-targeted core shell-like assemblies . This indicates that pEP84R directs the co-assembly of these polyproteins at the correct cellular location.

• Hierarchical relationship: Research suggests a hierarchical relationship where pEP84R acts upstream of pp220 and pp62 in the assembly pathway. pEP84R first localizes to the ER membrane, then recruits pp220 through direct binding, which subsequently facilitates pp62 incorporation through pp220-pp62 interactions .

• Functional interdependence: The proper function of all three proteins is interdependent. While pEP84R guides the localization of pp220 and pp62, these polyproteins are essential for core shell formation. Neither component alone is sufficient for proper assembly .

This relationship represents a key regulatory mechanism in ASFV morphogenesis, ensuring that core assembly occurs at the correct location and in the correct sequence .

How might pEP84R serve as a target for antiviral development against ASFV?

The pEP84R protein presents a promising target for antiviral development against ASFV for several reasons:

• Essential function: pEP84R plays a crucial role in core assembly, and its absence results in non-infectious viral particles . Targeting this protein could effectively block viral replication.

• Unique mechanism: The protein's specific role in guiding core assembly represents a unique mechanism that could be targeted without affecting host cellular processes .

• Potential antiviral strategies:

  • Small molecule inhibitors: Compounds that bind to pEP84R and prevent its interaction with pp220

  • Peptide-based inhibitors: Designed to mimic the N-terminal region of pp220 and competitively inhibit its binding to pEP84R

  • RNA interference: siRNAs targeting EP84R mRNA to reduce protein expression

  • CRISPR-based approaches: For genetically modifying the virus in vaccine development

• Conserved target: If the EP84R gene is conserved across ASFV strains, therapeutics targeting this protein could potentially be effective against multiple virus variants .

Research indicates that these findings "unveil a key regulatory mechanism for ASFV morphogenesis and identify a relevant novel target for the development of therapeutic tools against this re-emerging threat" , highlighting the potential of pEP84R-targeted approaches in combating ASFV.

What considerations should be made when designing attenuated vaccines based on EP84R modification?

When designing attenuated vaccines based on EP84R modification, researchers should consider several critical factors:

• Attenuation strategy:

  • Conditional expression: Creating viruses with inducible EP84R expression similar to vEP84Ri

  • Partial deletions: Removing specific functional domains rather than the entire protein

  • Point mutations: Introducing mutations that reduce but don't eliminate function

• Safety considerations:

  • Reversion potential: Assessing the likelihood of mutations that could restore virulence

  • Residual virulence: Testing thoroughly in animal models before field application

  • Genetic stability: Ensuring modifications remain stable over multiple viral generations

• Immunogenicity assessment:

  • Protective antigen presence: Verifying that key protective antigens are still expressed

  • Immune response profile: Characterizing both humoral and cell-mediated responses

  • Protection efficacy: Testing against challenge with virulent ASFV strains

• Technical hurdles:

  • Growth in culture: Modified viruses must grow to sufficient titers for vaccine production

  • Genetic verification: Confirming the intended modifications remain intact

  • Differentiating infected from vaccinated animals (DIVA): Incorporating markers to distinguish vaccine strains from field strains

Table 2: Potential EP84R Modification Strategies for Vaccine Development

How does the structure-function relationship of pEP84R compare across different ASFV strains?

The structure-function relationship of pEP84R across different ASFV strains presents an area that requires deeper investigation, though existing research provides some insights:

Future research using comparative genomics, structural biology approaches, and functional assays with pEP84R from diverse ASFV isolates would help establish the evolutionary constraints on this protein and potentially identify strain-specific therapeutic targets .

What methodological challenges exist in studying the temporal dynamics of pEP84R during viral assembly?

Studying the temporal dynamics of pEP84R during viral assembly presents several methodological challenges:

• Real-time visualization limitations:

  • Difficulty in distinguishing between different assembly intermediates

  • Limited resolution of conventional light microscopy for small viral components

  • Potential artifacts when using fluorescently-tagged proteins

  • Challenge of maintaining viral activity during live imaging

• Synchronization of infection:

  • ASFV replication is not perfectly synchronized in cell culture

  • Difficult to isolate specific assembly stages for biochemical analysis

  • Various assembly steps may occur simultaneously in different cellular regions

• Complex interactions network:

  • pEP84R likely participates in multiple protein-protein interactions beyond pp220

  • Teasing apart temporal sequence of interactions requires sophisticated approaches

  • May involve transient interactions difficult to capture with standard techniques

• Technical approaches to overcome these challenges:

  • Super-resolution microscopy techniques (STORM, PALM)

  • Correlative light and electron microscopy (CLEM)

  • Pulse-chase experiments with inducible systems

  • Cross-linking mass spectrometry to capture transient interactions

  • Single-particle tracking in live cells

  • Cryo-electron tomography of assembly intermediates

Advanced time-resolved approaches that combine multiple techniques will be necessary to fully understand the dynamic role of pEP84R throughout the ASFV assembly process .

How might computational modeling help predict the effects of specific mutations in pEP84R on ASFV assembly?

Computational modeling offers powerful approaches to predict how specific mutations in pEP84R might affect ASFV assembly:

• Structural prediction and modeling:

  • Homology modeling to predict the three-dimensional structure of pEP84R

  • Molecular dynamics simulations to understand protein flexibility and movement

  • Prediction of transmembrane domain orientation and membrane insertion

  • Modeling of protein-protein interfaces between pEP84R and pp220

• Mutation effect prediction:

  • In silico mutagenesis to assess the impact of specific amino acid substitutions

  • Calculation of changes in binding affinity with partner proteins

  • Prediction of altered membrane interactions for transmembrane domain mutations

  • Identification of critical residues through evolutionary conservation analysis

• Systems-level modeling:

  • Agent-based models of the assembly process to predict emergent behaviors

  • Kinetic models of assembly to identify rate-limiting steps

  • Prediction of how mutations might alter assembly pathways or kinetics

  • Integration of spatial and temporal aspects of viral assembly

• Validation approaches:

  • Experimental testing of computational predictions using site-directed mutagenesis

  • Correlation of predicted structural changes with observed phenotypes

  • Iterative refinement of models based on experimental feedback

Such computational approaches could significantly accelerate the identification of critical functional regions in pEP84R and guide rational design of mutations for attenuated vaccines or antiviral targets . These methods also help generate testable hypotheses about the molecular mechanisms underlying pEP84R function in ASFV assembly.

What are the most promising future research directions regarding pEP84R?

The study of pEP84R has opened several promising research avenues that could advance our understanding of ASFV biology and lead to effective control strategies:

• Structural biology: Determining the high-resolution structure of pEP84R, particularly in complex with pp220, would provide crucial insights into their interaction mechanism and guide structure-based drug design .

• Comprehensive interactome mapping: Identifying all viral and cellular proteins that interact with pEP84R could reveal additional roles beyond core assembly and potential new therapeutic targets .

• Cross-strain functional analysis: Comparative studies of pEP84R from different ASFV isolates could identify conserved functional domains and strain-specific variations that might correlate with virulence differences .

• Rational attenuated vaccine development: Engineering specific mutations in EP84R to create stable attenuated ASFV strains with potential vaccine applications .

• Small molecule inhibitor screening: High-throughput screening for compounds that disrupt the pEP84R-pp220 interaction could yield candidate antivirals .

• Temporal assembly dynamics: Detailed investigation of the sequence and timing of pEP84R's role during viral morphogenesis using advanced imaging techniques .

• Host factors involved in pEP84R function: Identifying cellular components that facilitate or regulate pEP84R's activity could provide alternative therapeutic targets .

These research directions collectively represent a comprehensive approach to understanding and exploiting pEP84R's critical role in ASFV biology for scientific and practical applications.

How does the study of pEP84R contribute to our broader understanding of complex virus assembly?

The characterization of pEP84R contributes significantly to our broader understanding of complex virus assembly mechanisms:

• Model for multilayered virus morphogenesis: ASFV's complex structure provides a unique model for understanding how large DNA viruses coordinate the assembly of multiple structural layers. pEP84R's role illustrates how transmembrane proteins can guide this process .

• Membrane-associated virus assembly: The study of pEP84R reveals mechanisms by which viruses utilize cellular membranes as assembly platforms, informing our understanding of similar processes in other enveloped viruses .

• Hierarchical assembly coordination: pEP84R exemplifies how viruses employ regulatory proteins to ensure the correct spatial and temporal sequence of assembly events, a principle likely applicable across various virus families .

• Polyprotein processing and assembly: The interaction between pEP84R and viral polyproteins provides insights into how processing of polyprotein precursors is coordinated with structural assembly .

• Nucleocapsid formation mechanisms: The role of pEP84R in enabling genome packaging illustrates fundamental principles of how virus cores are formed around genetic material .

• Protein targeting in viral factories: pEP84R's function in directing core components to specific cellular locations enhances our understanding of how viruses create specialized replication compartments .