Recombinant African swine fever virus Inner membrane protein p12

What is the confirmed localization of ASFV p12 protein within the virion structure?

Notably, a detailed morphogenesis study found that "p12 localizes at the perinuclear virus factories as well as into virus particles spread throughout the cytoplasm and the cell surface," with a subcellular distribution similar to the inner envelope protein p17 . Furthermore, immunoelectron microscopy on thawed cryosections of infected cells showed p12 is associated with viral membrane precursors, assembling particles, and intracellular mature viruses .

The discrepancy in localization findings highlights the complexity of ASFV's multilayered structure, which includes the envelope, capsid, inner envelope, core shell, and nucleoid from outside to inside .

How does p12 contribute to ASFV entry into host cells?

p12 plays a crucial role in the initial stages of ASFV infection by mediating virus attachment to host cells. Previous studies demonstrated that:

• p12 mediates the binding of ASFV to permissive cells in both saturable and nonsaturable interactions

• It allows membrane proteins on the cell surface to act as ASFV receptors

• Treatment of cell surfaces with proteases inhibits p12 binding activity, while glycosidases or lipase treatments do not affect binding, suggesting the receptor is primarily protein-based with no significant involvement of carbohydrates or lipids

The virus entry process involves multiple proteins, including p12, p54, p72, and CD2v, which collaborate to facilitate ASFV adsorption onto host cells . The complete entry mechanism involves a complex sequence where viral particles are internalized through both constitutive macropinocytosis and clathrin-mediated endocytosis, followed by movement to late multivesicular endosomes where uncoating occurs .

What is known about the molecular structure of p12?

Based on available research, p12 is characterized as:

• A transmembrane protein located at the inner envelope of the ASFV structure

• Involved in virus morphogenesis

• Contains specific epitopes that can induce antibody production in infected animals

What methods are most effective for producing recombinant p12 protein?

Several approaches have been successfully employed to produce recombinant p12 protein:

• Bacterial expression systems: Recombinant p12 has been successfully expressed in E. coli, as evidenced by the available commercial recombinant p12 protein (amino acids 1-62) derived from the Pig/Kenya/KEN-50/1950 isolate . This approach typically involves:

  • Cloning the p12 gene into an appropriate expression vector (e.g., pET28a)

  • Transforming the construct into an E. coli expression strain

  • Inducing protein expression with IPTG

  • Purifying the protein using affinity chromatography methods

• Synthetic genomics-based approaches: Recent advances in ASFV reverse genetics systems enable the production of modified viruses with altered p12 genes, allowing study of the protein in its native context . This method involves:

  • In vitro CRISPR-Cas9 editing of ASFV TAR clones

  • Assembly of modified viral genomes in yeast and E. coli

  • Transfection of the assembled genome into permissive cells

  • Recovery and characterization of recombinant viruses

• Epitope-based approaches: For immunological studies, researchers have produced recombinant antigenic proteins containing multiple epitopes, including those from p12, as part of vaccine development efforts . This method involves:

  • Identifying conserved B-cell epitopes across multiple strains

  • Designing synthetic constructs with selected epitopes

  • Expression in appropriate systems with adjuvants

  • Validation through immunogenicity testing

What assays can be used to study p12's interaction with host cell receptors?

Based on the research methodologies described in the search results, several assays have been used to study p12-receptor interactions:

• Binding assays with recombinant p12:

  • Using purified recombinant p12 to study binding to different cell lines

  • Analyzing saturable and nonsaturable binding kinetics

  • Quantifying binding through fluorescently labeled proteins or antibodies

• Enzyme treatment studies:

  • Treating cell surfaces with different enzymes (proteases, glycosidases, lipases)

  • Measuring the effect on p12 binding to identify the biochemical nature of cellular receptors

• Immunological techniques:

  • Using anti-p12 antibodies to block virus binding to cells

  • Evaluating whether neutralizing antibodies against p12 inhibit virus infection

• Virus attachment assays:

  • Comparing binding of wild-type virus versus p12-deleted or modified viruses

  • Using flow cytometry in combination with pharmacological entry inhibitors

  • Fluorescence and electron microscopy approaches to visualize virus-cell interactions

How can researchers accurately assess p12 localization within the ASFV virion?

Given the contradictory findings regarding p12 localization, researchers should employ multiple complementary techniques:

• Immunoelectron microscopy:

  • Prepare thawed cryosections of infected cells or purified virions

  • Label with specific anti-p12 antibodies followed by gold-conjugated secondary antibodies

  • Analyze localization relative to other viral structural proteins with known positions

• Confocal immunofluorescence microscopy:

  • Perform co-localization studies with markers for different viral compartments

  • Compare p12 distribution with known inner envelope proteins (e.g., p17) and outer envelope proteins

• Biochemical fractionation:

  • Isolate and fractionate viral particles into their constituent layers

  • Analyze the presence of p12 in different fractions using Western blotting

  • Compare with the distribution pattern of proteins with confirmed localization

• Cryo-electron tomography:

  • Obtain high-resolution 3D reconstructions of intact virions

  • Use immunogold labeling to map p12 localization

  • Correlate with known structural features of the virus

How does p12 function within the context of ASFV's complex membrane fusion mechanism?

The entry process of ASFV involves a sophisticated mechanism where the virus undergoes uncoating in late multivesicular endosomes, followed by fusion of the inner viral membrane with the endosomal membrane. Current research suggests:

• The inner envelope proteins, including potentially p12, play critical roles in fusion events

• Virus uncoating requires acidic pH and involves disruption of the outer membrane and protein capsid

• The inner viral membrane becomes exposed and fuses with the limiting endosomal membrane

• This fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with members of the poxviral entry/fusion complex

• Another inner membrane protein, pE199L, has recently been shown to be required for membrane fusion and core penetration, indicating it works together with pE248R as part of ASFV's fusion machinery

The exact role of p12 in this process remains to be fully elucidated, but its localization to the inner envelope suggests it may interact with other fusion-related proteins like pE248R and pE199L. A comprehensive study of these interactions would require co-immunoprecipitation experiments, proximity labeling approaches, or structural studies of the protein complex.

What is the relationship between p12 and other viral structural proteins during ASFV assembly?

ASFV assembly is a complex process occurring in virus factories (VFs) at the host perinuclear region. The relationship between p12 and other viral proteins during assembly involves:

• p12 localizes at virus factories during assembly, showing a distribution pattern similar to the inner envelope protein p17

• Immunoelectron microscopy indicates p12 associates with viral membrane precursors and assembling particles

• The assembly process involves multiple steps where the core shell proteins are deposited on membrane assembly intermediates

• Other proteins like pA104R, pp220, and pp62 are involved in genome packaging and core formation

• The viral proteolytic enzyme pS273R catalyzes processing of polyprotein precursors during assembly

To better understand p12's specific interactions during assembly, researchers could employ:

• Proximity-dependent biotinylation approaches to identify proteins in close contact with p12

• Time-course imaging studies to track p12 localization during different stages of virus assembly

• Genetic approaches using inducible expression systems to determine whether p12 is required for correct localization of other viral structural proteins

What's the current understanding of p12 conservation across different ASFV genotypes?

The conservation of p12 across ASFV genotypes is an important consideration for both fundamental virology research and vaccine development. While the search results don't provide specific sequence conservation data for p12, they offer relevant context:

• ASFV has been classified into 24 genotypes based on the p72 gene sequence

• This genotype diversity presents challenges for developing broadly protective vaccines

• Recent approaches for vaccine development have focused on identifying conserved B-cell epitopes across multiple strains, including those from p12

To comprehensively analyze p12 conservation, researchers should:

• Perform multiple sequence alignments of p12 sequences from all available ASFV genotypes

• Calculate sequence identity/similarity percentages and identify conserved regions

• Map conservation data onto structural models (if available) to identify surface-exposed conserved regions

• Analyze whether conserved regions correspond to functionally important domains

This information would be valuable for understanding the evolutionary constraints on p12 and evaluating its potential as a broadly protective antigen for vaccine development.

How do researchers explain the contradictory findings regarding p12 localization in the viral particle?

The contradictory findings regarding p12 localization (outer envelope versus inner envelope) might be explained by several factors:

• Methodological differences:

  • Earlier studies suggesting outer envelope localization might have used different detection methods or sample preparation techniques compared to later studies

  • The complex structure of ASFV might lead to different results depending on fixation, embedding, and sectioning methods used for electron microscopy

• Dynamic distribution:

  • p12 might redistribute during virus maturation and egress

  • It could be present in both membranes but at different concentrations or conformations

  • Its exposure or accessibility to antibodies might change during different stages of the viral life cycle

• Strain differences:

  • Different ASFV isolates might have variations in p12 localization or expression

  • Adaptations to cell culture might affect protein localization

A systematic study combining multiple localization techniques (immunogold EM, super-resolution microscopy, biochemical fractionation) across different ASFV strains and at different stages of infection would help resolve this contradiction.

What aspects of p12 structure-function relationship remain poorly understood?

Despite progress in understanding p12's role in ASFV infection, several knowledge gaps remain:

• Atomic-level structure:

  • The three-dimensional structure of p12 has not been reported

  • Without structural information, it's difficult to understand how p12 interacts with cellular receptors or other viral proteins at the molecular level

• Specific binding partners:

  • The cellular receptor(s) that p12 binds to remain unidentified

  • The exact protein-protein interactions between p12 and other viral components are not well characterized

• Fusion mechanism involvement:

  • Whether p12 participates directly in membrane fusion events during entry is unclear

  • Its potential interaction with confirmed fusion proteins like pE248R and pE199L needs investigation

• Post-translational modifications:

  • Information about potential glycosylation, phosphorylation, or other modifications of p12 is limited

  • How such modifications might affect p12 function remains unknown

Research approaches to address these gaps could include structural biology techniques (X-ray crystallography, cryo-EM), systematic protein-protein interaction studies, and targeted mutagenesis of p12 combined with functional assays.

How effective are p12-based recombinant subunit vaccines against ASFV?

For developing effective p12-based vaccines, researchers should consider:

• Combining p12 epitopes with other immunogenic ASFV proteins

• Using appropriate adjuvants to enhance immune responses

• Evaluating protection against different ASFV genotypes

• Assessing both humoral and cell-mediated immune responses

The available data suggests that while p12 may be antigenic, it might be most effective as one component of a multi-epitope or multi-protein vaccine strategy rather than as a standalone antigen.

What methodologies can be used to develop CRISPR-Cas9 modified ASFV with altered p12 for vaccine research?

The development of modified ASFV strains with altered p12 can be achieved using innovative genome engineering approaches, as described in the search results:

• Synthetic genomics-based reverse genetics system:

  • This approach enables the generation of recombinant ASFV mutant strains with modifications across the genome

  • The method involves:
    a) Fragmenting the ASFV genome into manageable pieces
    b) Modifying specific fragments using in vitro CRISPR-Cas9 editing
    c) Assembling modified fragments in yeast and E. coli
    d) Transfecting assembled genomes into WSL-gRO61R cells infected with helper virus
    e) Selecting recombinant viruses using fluorescent markers and Cas9-resistant modifications

• Considerations for p12 modification:

  • The p12 gene has been successfully targeted in this system, with modifications making it resistant to Cas9 cleavage

  • This approach could be extended to create p12 deletion mutants, epitope-tagged versions, or point mutations

  • Functional domains identified through structural or biochemical studies could be specifically targeted

• Evaluation of modified viruses:

  • Characterize growth kinetics and plaque formation of modified viruses

  • Assess virus binding and entry efficiency

  • Evaluate immunogenicity and protective efficacy in appropriate animal models

  • Test cross-protection against heterologous ASFV strains

This methodology provides powerful tools for studying p12 function in context and for rational design of attenuated vaccine candidates.

How might understanding p12 function lead to novel antiviral strategies against ASFV?

Understanding p12's role in ASFV infection could inform several antiviral strategies:

• Entry inhibitors:

  • If p12 is indeed involved in virus attachment, small molecules or peptides that bind p12 could block viral entry

  • Soluble receptor mimics could compete with cellular receptors for p12 binding

  • Structure-based drug design targeting p12 would require solving its three-dimensional structure

• Host-targeted approaches:

  • Identifying the cellular receptor(s) for p12 could lead to strategies that transiently downregulate or block these receptors

  • Understanding the signaling pathways activated upon p12-receptor interaction might reveal additional targets

• Fusion inhibitors:

  • If p12 cooperates with pE248R and pE199L in membrane fusion, compounds disrupting this interaction could block viral penetration

  • This approach would be similar to fusion inhibitors used against other viruses like HIV

• Broadly protective vaccines:

  • Identifying conserved, functionally crucial epitopes in p12 could guide the development of vaccines effective against multiple ASFV genotypes

  • These epitopes could be incorporated into multi-epitope vaccines or used to design immunogens that elicit broadly neutralizing antibodies

The development of a recombinant ASFV RNA polymerase system for antiviral drug screening in low biosafety containment environments provides a model for how mechanistic understanding of viral proteins can lead to practical applications in antiviral discovery.

Table 1: Key ASFV Structural Proteins and Their Functions

Table 2: Experimental Approaches for Studying ASFV p12