Recombinant African swine fever virus Uncharacterized membrane protein KP93L (War-001)

What is KP93L/War-001 protein and what do we know about its basic characteristics?

KP93L (War-001) is an uncharacterized membrane protein encoded by the African Swine Fever Virus. It was initially identified in the Warthog/Namibia/Wart80/1980 isolate (UniProt ID: P0CAL9) . The protein is located in the terminal inverted repeat (TIR) region of the ASFV genome, and interestingly, in some ASFV strains, this gene and its counterpart DP93R are perfectly repeated but inverted at opposite ends of the viral genome .

The full-length recombinant protein consists of 68-93 amino acids (differences in length have been reported between isolates), with the BA71V strain variant having 93 amino acids with the sequence: MFFFGIFRCNMDHWTTKRQVYIYLCFSLMTIALICYLIHICCHTKKNVVTNALPSNNMALIPYTPSNNMALIPYTPSNNTVPPPYTISGSCPQ . Its membrane-associated nature suggests potential roles in virus-host interactions, though its specific functions remain largely undefined.

How does the genetic structure of KP93L differ across ASFV strains?

The KP93L gene exhibits notable structural variations across different ASFV isolates. In BA71V (an attenuated laboratory strain), there exists a block of three RTD33 direct tandem repeats that originated from the duplication of one of the two RDT33 repeats found in the virulent BA71 strain . This variation affects the KP93L/DP93R genes, resulting in a repetition of 11 amino acids near the C-terminus of the encoded protein in BA71V .

All European isolates whose genome sequences include the TIR region maintain the BA71 structure rather than the BA71V structure . This suggests that the duplication event in BA71V might be related to laboratory adaptation or attenuation. The structural variation in KP93L across virulent field isolates versus attenuated laboratory strains provides an important research avenue for understanding ASFV virulence mechanisms.

Are there any known host interaction partners for KP93L?

To identify potential interaction partners, researchers would need to employ techniques such as co-immunoprecipitation followed by mass spectrometry, yeast two-hybrid screening, or proximity labeling approaches (BioID or APEX). The recombinant His-tagged version of KP93L available commercially could serve as a valuable tool for such interaction studies, enabling pull-down experiments with potential binding partners from host cell lysates .

How might KP93L contribute to ASFV virulence or attenuation?

The potential role of KP93L in ASFV virulence remains an open research question. Interestingly, the structural differences in KP93L between the virulent BA71 strain and its attenuated derivative BA71V suggest possible implications for virulence. In BA71V, KP93L contains a duplication resulting in three RTD33 repeats instead of the two found in BA71, leading to an 11-amino acid repetition near the C-terminus .

Experimental approaches to investigate KP93L's role in virulence could include:

• Creating recombinant viruses with KP93L deletions or modifications

• Comparing the properties of KP93L between virulent and attenuated strains

• Analyzing the impact of KP93L on host immune responses

What structural and functional domains exist within KP93L and what are their predicted roles?

Analysis of the KP93L amino acid sequence (MFFFGIFRCNMDHWTTKRQVYIYLCFSLMTIALICYLIHICCHTKKNVVTNALPSNNMALIPYTPSNNMALIPYTPSNNTVPPPYTISGSCPQ) reveals several features that may relate to its function :

• The N-terminal region appears hydrophobic (MFFFGIFRC...), suggesting potential membrane insertion or association.

• The protein contains multiple repeated sequences (e.g., "NNMALIPYT" appears twice), which may be involved in protein-protein interactions or structural stability.

• The C-terminal region shows repeated proline residues (PPPY), which might form a polyproline type II helix, often involved in protein-protein interactions.

Functional predictions based on sequence analysis suggest KP93L might play roles in:

• Membrane organization or modification during infection

• Scaffolding for viral assembly

• Modulation of host cell processes through protein-protein interactions

Advanced structural studies using X-ray crystallography or NMR would be necessary to definitively characterize these domains and their functions.

What experimental approaches are most effective for studying the function of an uncharacterized viral protein like KP93L?

Investigating an uncharacterized viral protein like KP93L requires a multi-faceted approach:

• Genetic manipulation studies:

  • CRISPR-Cas9 editing of the viral genome to create KP93L deletion mutants

  • Site-directed mutagenesis to modify potential functional domains

  • Complementation studies to restore function in deletion mutants

• Protein localization and dynamics:

  • Fluorescent protein tagging for live-cell imaging

  • Immunofluorescence with specific antibodies against KP93L

  • Subcellular fractionation followed by western blotting

• Protein-protein interaction studies:

  • Co-immunoprecipitation with suspected interaction partners

  • Proximity labeling (BioID, APEX)

  • Yeast two-hybrid screening

• Functional assays:

  • Impact on viral replication kinetics

  • Effects on host cell pathways (apoptosis, immune signaling)

  • Membrane permeability and integrity assessment

• Structural biology approaches:

  • X-ray crystallography or cryo-EM for 3D structure determination

  • NMR for dynamic structural information

  • Molecular dynamics simulations based on predicted structure

How might the genomic location of KP93L in the terminal inverted repeat (TIR) region influence its evolution and function?

The location of KP93L in the terminal inverted repeat (TIR) region of the ASFV genome has significant implications for its evolution and function. In the BA71V strain, KP93L and its counterpart DP93R are located at opposite ends of the genome in an inverted orientation . This genomic architecture suggests several important considerations:

• Evolutionary implications:

  • The duplication and inversion event creating these paired genes likely resulted from recombination events during viral evolution

  • The presence of these genes in the highly variable terminal regions suggests they may be under different selective pressures than core viral genes

  • Changes in one copy might be compensated by the other copy, potentially allowing for more rapid evolution

• Functional considerations:

  • Terminal genes often play roles in viral DNA replication or genome packaging

  • The inverted repeat structure may facilitate circularization of the genome during replication

  • The presence of repetitive elements (RTD33) within these genes suggests potential roles in genomic plasticity or adaptation

• Research approaches:

  • Comparative genomics across multiple ASFV isolates to track KP93L/DP93R evolution

  • Analysis of selection pressures on these genes compared to core viral genes

  • Experimental deletion of one or both copies to determine functional redundancy

What are the optimal conditions for expressing and purifying recombinant KP93L protein?

Recombinant KP93L protein expression and purification has been successfully accomplished using the following methodology:

• Expression system: E. coli has proven effective for expressing recombinant KP93L with N-terminal His-tag .

• Purification strategy:

  • Metal affinity chromatography using the His-tag

  • Follow with size exclusion chromatography for higher purity if needed

• Storage conditions:

  • The purified protein is typically prepared as a lyophilized powder

  • Recommended reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Addition of 5-50% glycerol (final concentration) is advised for long-term storage

  • Storage at -20°C/-80°C, avoiding repeated freeze-thaw cycles

• Buffer considerations:

  • Tris/PBS-based buffer, pH 8.0, with 6% Trehalose has been used successfully

  • For membrane proteins like KP93L, addition of mild detergents may improve solubility and stability

• Quality control:

  • SDS-PAGE analysis typically shows >90% purity

  • Western blotting with anti-His antibodies confirms identity

  • Mass spectrometry can verify the correct sequence

What techniques are most appropriate for studying the membrane association and topology of KP93L?

As KP93L is described as a membrane protein, specialized techniques are required to study its membrane association and topology:

• Computational prediction approaches:

  • Transmembrane domain prediction using algorithms like TMHMM, Phobius, or TOPCONS

  • Hydropathy plot analysis to identify potential membrane-spanning regions

  • Signal peptide prediction to determine membrane insertion mechanisms

• Biochemical approaches:

  • Membrane fractionation to confirm association with cellular membranes

  • Protease protection assays to determine topology (which regions face cytoplasm vs. lumen)

  • Chemical labeling of accessible cysteine residues before and after membrane permeabilization

• Structural biology approaches:

  • Cryo-electron microscopy of KP93L in lipid nanodiscs or liposomes

  • Solid-state NMR spectroscopy for membrane-embedded proteins

  • Hydrogen-deuterium exchange mass spectrometry to map solvent-accessible regions

• Fluorescence-based approaches:

  • FRET analysis with fluorophores positioned at different locations

  • GFP-fusion analysis with split-GFP complementation to determine topology

  • Fluorescence microscopy to determine subcellular localization in infected cells

How can researchers generate and validate specific antibodies against KP93L for immunological studies?

Developing specific antibodies against KP93L requires careful planning and validation:

• Antigen design strategies:

  • Use full-length recombinant KP93L protein for immunization

  • Alternatively, design synthetic peptides based on predicted epitopes, particularly from hydrophilic regions

  • Consider multiple peptides from different regions of the protein for increased specificity

• Antibody production approaches:

  • Polyclonal antibodies: Immunize rabbits or guinea pigs with purified recombinant KP93L

  • Monoclonal antibodies: Screen hybridoma supernatants for specific recognition of KP93L

  • Recombinant antibodies: Phage display selection against purified KP93L

• Validation methods:

  • Western blotting against recombinant protein and viral lysates

  • Immunoprecipitation followed by mass spectrometry

  • Immunofluorescence in infected versus uninfected cells

  • Peptide competition assays to confirm specificity

  • Testing in KP93L knockout/knockdown systems as negative controls

• Applications:

  • Localization studies during infection

  • Co-immunoprecipitation to identify interaction partners

  • ChIP-seq if there's evidence of DNA-binding activity

  • ELISA development for detecting viral infection

What functional assays can be developed to investigate the role of KP93L during ASFV infection?

To investigate the functional role of KP93L during ASFV infection, several specialized assays can be developed:

• Viral genetics approaches:

  • Generate KP93L deletion mutants using CRISPR-Cas9 or homologous recombination

  • Create point mutations in predicted functional domains

  • Develop complementation systems to restore KP93L function

• Infection dynamics assays:

  • Growth curve analysis comparing wild-type and KP93L-mutant viruses

  • Single-step and multi-step growth curves in different cell types

  • Plaque size and morphology assessment

• Host-interaction assays:

  • RNA-seq to compare host transcriptional responses to wild-type vs. KP93L-mutant viruses

  • Proteomics to identify changes in host protein abundance or post-translational modifications

  • Reporter assays for key host signaling pathways (NF-κB, IRF3, etc.)

• Cellular process impact:

  • Membrane integrity and permeability assays

  • Apoptosis and cell death measurements

  • Cytokine production and immune signaling assessment

  • Autophagy and cellular stress response monitoring

• In vivo studies:

  • Pathogenesis studies comparing wild-type and KP93L-mutant viruses in natural hosts

  • Immune response characterization in infected animals

  • Transmission studies to assess impact on viral spread

What are the major knowledge gaps in our understanding of KP93L function?

Despite the availability of recombinant KP93L protein and genomic data, significant knowledge gaps remain:

• The precise molecular function of KP93L remains unknown, as it is still classified as an "uncharacterized membrane protein" .

• The significance of the structural variations in KP93L between virulent strains (like BA71) and attenuated strains (like BA71V) has not been fully elucidated .

• The temporal expression pattern of KP93L during infection has not been specifically characterized, though methodologies exist for such analysis .

• The contribution of KP93L to viral replication, assembly, or host immune evasion remains to be determined.

• The structure-function relationship of the repeated elements within KP93L sequence requires investigation .

How might comparative analysis between different ASFV strains inform KP93L research?

Comparative analysis between different ASFV strains offers valuable insights for KP93L research:

• Sequence conservation and variation:

  • The BA71 and BA71V strains show key differences in KP93L structure, with BA71V having three RTD33 repeats compared to two in BA71

  • European isolates maintain the BA71 structure rather than the BA71V structure

  • Analyzing these variations across multiple isolates could reveal evolutionary patterns

• Structure-function correlations:

  • Comparing KP93L sequences from virulent field isolates versus attenuated laboratory strains

  • Correlating specific sequence features with phenotypic differences

  • Examining whether changes in KP93L correlate with host range or tissue tropism

• Experimental approaches:

  • Chimeric virus construction swapping KP93L variants between strains

  • Site-directed mutagenesis to introduce specific variations observed across strains

  • Infection studies comparing viruses with different KP93L variants

How might systems biology approaches enhance our understanding of KP93L in the context of ASFV pathogenesis?

Systems biology approaches offer powerful tools for understanding KP93L's role in ASFV pathogenesis:

• Integrative omics approaches:

  • Combine transcriptomics, proteomics, and metabolomics data from ASFV infections

  • Compare wild-type virus with KP93L mutants to identify affected pathways

  • Integrate host and viral data to build comprehensive interaction networks

• Mathematical modeling:

  • Develop predictive models of ASFV infection incorporating KP93L functions

  • Simulate the effects of KP93L mutations on viral replication dynamics

  • Model host-pathogen interactions at different scales (molecular to organismal)

• Network analysis:

  • Construct protein-protein interaction networks centered on KP93L

  • Identify key host pathways potentially disrupted by KP93L

  • Predict functional roles based on network positioning

• Single-cell approaches:

  • Single-cell RNA-seq to capture cell-to-cell variability in response to KP93L

  • Spatial transcriptomics to map infection patterns in tissues

  • Correlate KP93L expression with cellular outcomes

What are the key considerations for researchers beginning work with KP93L?

Researchers initiating studies on KP93L should consider:

• Starting materials: Recombinant KP93L protein is commercially available as a His-tagged construct expressed in E. coli, which provides a valuable starting point for structural and functional studies .

• Strain selection: Choose appropriate ASFV strains for comparison, considering that KP93L shows structural variations between virulent field isolates (like BA71) and attenuated laboratory strains (like BA71V) .

• Methodological approach: Employ both computational predictions and experimental validations when studying this uncharacterized membrane protein.

• Integration with other ASFV research: Consider KP93L in the broader context of ASFV biology, particularly its potential relationships with virulence factors and host interactions.

• Technical challenges: Prepare for the challenges inherent in studying membrane proteins, including issues with solubility, purification, and structural analysis.

What research priorities should guide future investigations of KP93L?

Future KP93L research should prioritize:

• Functional characterization: Determine the precise molecular function of KP93L through targeted mutational analysis, host-interaction studies, and functional assays.

• Structural biology: Resolve the three-dimensional structure of KP93L to understand how its sequence features, particularly the repeated elements, contribute to its function.

• Role in viral lifecycle: Characterize KP93L's temporal expression pattern and localization during infection to understand its role in the viral lifecycle.

• Contribution to virulence: Investigate whether variations in KP93L structure between virulent and attenuated strains contribute to differences in pathogenicity.

• Potential as intervention target: Assess whether KP93L could serve as a target for antiviral interventions or as a component of attenuated vaccine designs.