Recombinant African swine fever virus Uncharacterized protein B117L (War-093)
Description
Introduction to African Swine Fever Virus and the B117L Protein
African swine fever virus is a devastating pathogen causing a pandemic that has resulted in significant economic losses in the swine industry across Central Europe to East Asia. The virus contains a large double-stranded DNA genome of approximately 170-193 kilobases encoding more than 150 genes, most with previously uncharacterized functions . Among these, the B117L gene has recently garnered attention for its role in viral replication and cell entry.
The B117L gene encodes a small 115-amino-acid integral membrane protein that is transcribed late during the virus replication cycle and shows no homology to any previously published protein . The protein product has been identified as having viroporin-like activity, assisting in the permeabilization of the endoplasmic reticulum (ER)-derived envelope during ASFV infection . This discovery represents a significant advancement in understanding ASFV biology, as the insufficient knowledge regarding the function of most ASFV genes has historically limited the development of effective countermeasures against this pathogen.
Transmembrane Topology and Structure
Hydrophobicity distribution analysis confirmed the presence of a single transmembrane helix (TMH), which in combination with flanking amphipathic sequences, composes the membrane-associated C-terminal domain . Three-dimensional structure prediction using AlphaFold revealed a single helix structure (residues 67-114) that includes the TMH moiety .
The transmembrane helix traverses the membrane with an N-terminal out/C-terminal in topology, meaning the N-terminus is located in the lumen of the ER while the C-terminus faces the cytosol. This topology is dictated by the TMH section and is maintained even in the absence of a canonical signal peptide . The helix is preceded by a solvent-exposed small globular ectodomain at the N-terminus, while the membrane-spanning helix extends into the cytosol, exposing its C-terminal stretch (residues 90-114) to the solvent .
Temporal Expression During Viral Replication
Time course experiments analyzing RNA transcription kinetics in primary swine macrophages infected with ASFV strain Georgia (ASFV-G) revealed that B117L transcription was detected at 4 hours post-infection and remained stable until 24 hours post-infection . The expression pattern was similar to that of the late protein p72 (B646L), confirming that B117L is expressed late during the virus replication cycle .
Cellular Localization
When expressed in cells, B117L localizes to the endoplasmic reticulum (ER). Ectopic transient cell expression of the B117L gene as a green fluorescent protein (GFP) fusion protein revealed colocalization with markers of the ER . The protein adopts an N-terminal out/C-terminal in membrane topology, which is dictated by the transmembrane helix moiety .
Interestingly, some cells expressing the B117L-GFP construct displayed formation of organized smooth ER (OSER) structures, a phenomenon observed more frequently upon transfection with higher quantities of DNA . This ER restructuring was supported by the fact that the soluble ER marker BiP-mCherry appeared to be retained within the lumen of the formed structures, indicating that the ER seemed to collapse without membrane rupture .
Membrane Permeabilization Activity
One of the most significant findings regarding B117L is its ability to permeabilize membranes, particularly at low pH. Using partially overlapping peptides that encompassed the membrane domain sequence, researchers demonstrated that the B117L transmembrane helix has the capacity to establish spores and ion channels in membranes at low pH .
To test the possible pore-forming activity of B117L's membrane domain, three partially overlapping peptides (PB117L-1, PB117L-2, and PB117L-3) were designed and tested for their effects on membrane permeability using micrometer-sized giant unilamellar vesicles (GUVs) that mimicked the ER . The effects were compared at neutral and low pH, emulating conditions before and after entry of the ASFV through the endocytic route. The results showed that the GUV treated with PB117L-2 at pH 5.0 displayed membrane permeabilization, as evidenced by the internal volume of the GUV being marked with an externally added fluorescent probe .
Ion Channel Activity
Further experiments demonstrated that the B117L transmembrane domain can form ion channels in membranes. Unlike the proton-selective channels formed by influenza A virus M2 protein, the B117L pore-forming domain showed only weak discrimination for ions and small charged solutes . This ion channel activity was enhanced at low pH, consistent with a role in viral entry through the endocytic pathway .
Table 1: Comparison of Pore-Forming Activity Between B117L and Other Viral Proteins
| Protein | Virus | Channel Type | pH Sensitivity | Inhibitors | Function in Viral Life Cycle |
|---|---|---|---|---|---|
| B117L | ASFV | Weakly selective for ions and small solutes | Enhanced at low pH | Not inhibited by amantadine | Assists in permeabilization of ER-derived envelope during entry |
| M2 | Influenza A | Highly selective for protons | Activated at low pH | Inhibited by amantadine | Facilitates uncoating of viral particles during entry |
| B/M2 | Influenza B | Proton channel | Activated at low pH | Not inhibited by amantadine | Similar to Influenza A M2 |
| B169L | ASFV | Forms pores in ER-like membranes | Enhanced at low pH | Unknown | Potentially critical for production of infectious virus particles |
Role in Viral Entry
Based on the structural and functional characterization, B117L appears to play a crucial role in ASFV entry into host cells. The protein's ability to permeabilize membranes and form ion channels at low pH suggests that it assists in the permeabilization of the ER-derived envelope during ASFV infection . This viroporin-like activity is likely triggered at the low pH of the endosome during cell entry .
Attempts to delete the B117L gene from the genome of the parental ASFV strain Georgia were unsuccessful, suggesting that the function of B117L is essential for virus growth . This further supports the critical role of B117L in the viral life cycle.
Genetic Diversity and Conservation
Evolutionary analysis of the B117L gene revealed high conservation of the transmembrane domain during the evolution of the gene, indicating that the integrity of this domain is preserved by the action of purifying selection . This conservation suggests the importance of this domain for the function of the protein.
Natural Selection and Phenotypic Diversity
The B117L gene present in the isolate Ken05/Tk1 was found to represent a significantly different phenotype compared to other isolates, highlighting the role of natural selection in the evolution of different phenotypes . Despite these differences, the transmembrane domain remained highly conserved, suggesting that its function is critical for viral survival.
Expression Systems and Purification
Recombinant African swine fever virus Uncharacterized protein B117L (War-093) can be produced using in vitro Escherichia coli expression systems . The recombinant protein is typically tagged, often with an N-terminal 10xHis-tag, to facilitate purification . The expression region usually covers the full-length protein (amino acids 1-112 or 1-115, depending on the construct) .
Current Applications in ASFV Research
Recombinant B117L protein is primarily used for research purposes to understand the structure, function, and role of this protein in ASFV infection. Specific applications include:
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Structural studies to elucidate the three-dimensional arrangement of the protein
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Functional assays to characterize the membrane permeabilization and ion channel activities
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Evolutionary analyses to understand conservation patterns and selective pressures
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Immunological studies to investigate potential antigenic properties
Potential for Vaccine Development
Given the essential role of B117L in ASFV replication and its conservation across different isolates, this protein represents a potential target for vaccine development. Research on recombinant B117L could contribute to the development of subunit vaccines or other novel countermeasures against ASFV .
Future Research Directions
Several areas for future research on B117L include:
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Further structural characterization using high-resolution techniques like cryo-electron microscopy
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Detailed analysis of the ion channel properties to understand selectivity and gating mechanisms
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Investigation of potential inhibitors that could block the viroporin-like activity
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Exploration of the protein's interactions with other viral and host proteins
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Comparative studies with other viroporins to understand common mechanisms and differences
Product Specs
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Target Background
Q&A
What is ASFV B117L protein and what is its basic structural organization?
B117L is a 115-amino-acid integral membrane protein encoded by the ASFV genome. According to structural characterization studies, B117L contains a single transmembrane helix domain (residues 67-114). The 3D-structure prediction by AlphaFold revealed a configuration where the membrane-spanning helix is preceded by a solvent-exposed small globular ectodomain at the N-terminus. The C-terminal stretch (residues 90-114) extends into the cytosol, becoming exposed to solvent. The N-terminal strand that precedes the predicted transmembrane helix appears to contact the membrane interface at the external side . The protein has a predicted molecular weight of 25.7 kDa in its recombinant form .
What expression systems are commonly used for recombinant B117L production?
Recombinant B117L protein is primarily produced using Escherichia coli expression systems. Commercial sources and research laboratories utilize E. coli to generate the protein for experimental applications . After expression, the protein is typically purified to >90% purity as assessed by SDS-PAGE . The recombinant form of B117L is often produced with affinity tags (such as N-terminal His tags) to facilitate purification. For storage stability, the purified protein is commonly lyophilized in a buffer containing PBS (pH 7.4), 0.02% NLS, 1mM EDTA, 4% Trehalose, and 1% Mannitol .
How is B117L transcribed during the ASFV replication cycle?
B117L is transcribed early during ASFV infection. Transcriptome analysis using techniques such as mapping mRNA 5' ends has allowed researchers to identify the transcription start sites (TSSs) of ASFV genes, including B117L. In studies of ASFV temporal gene expression patterns, B117L belongs to the group of genes expressed during early infection . This early transcription timing suggests that B117L likely plays a role in the initial stages of viral replication rather than in later assembly or maturation processes.
What is the proposed function of B117L protein in the ASFV life cycle?
Recent experimental evidence indicates that B117L functions in the permeabilization of the endoplasmic reticulum (ER)-derived envelope during ASFV infection . This membrane permeabilization activity suggests that B117L plays a critical role in either viral entry or egress processes. The protein's transmembrane topology, with domains extending to both sides of the membrane, positions it ideally for modifying membrane permeability properties. This function could be crucial for the virus to breach cellular compartment barriers during its replication cycle .
How does B117L genetic diversity correlate with ASFV strain differences?
Studies on B117L genetic diversity have revealed significant variation among ASFV isolates. Research has identified specific B117L phenotypes that have evolved as a result of positive selection. For example, the B117L gene present in the ASFV isolate Ken05/Tk1 represents a significantly different phenotype compared to other isolates (P = 0.009 ≤ 0.05) . This genetic diversity may contribute to differences in virulence, host range, or immune evasion capabilities among ASFV strains, making B117L an important target for comparative genomic studies.
What experimental approaches can be used to study B117L protein-membrane interactions?
Several experimental approaches are valuable for investigating B117L interactions with cellular membranes:
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Membrane topology prediction and verification: Computational topology prediction followed by experimental verification using techniques like glycosylation mapping or protease protection assays.
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Lipid bilayer interactions: Reconstitution of purified B117L in artificial membrane systems such as POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayers to study direct effects on membrane integrity .
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Fluorescence-based permeabilization assays: Utilizing fluorescent dyes that change intensity upon membrane disruption to quantify B117L's permeabilization activity.
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Electrophysiology: Patch-clamp techniques to measure membrane conductance changes induced by B117L.
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Structural analyses: Techniques such as NMR spectroscopy or cryo-electron microscopy to visualize B117L-membrane interactions at molecular resolution.
How might B117L be targeted in vaccine development strategies?
B117L represents a potential target for ASFV vaccine development through several approaches:
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Deletion mutant vaccines: While B117L-specific deletion mutants haven't been prominently featured in the literature, similar approaches with other ASFV genes (such as I177L, MGF360/505 genes, and others) have shown promise in developing live-attenuated vaccines . The membrane permeabilization function of B117L suggests that deletion or modification might attenuate viral replication while maintaining immunogenicity.
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Subunit vaccines: Recombinant B117L protein could be included in subunit vaccine formulations, particularly if it contains conserved epitopes recognized by protective immune responses.
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Vector vaccines: The B117L gene could be incorporated into viral vector platforms to stimulate immune responses against this protein.
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Structure-based vaccine design: The defined membrane topology and structure of B117L could inform rational design of vaccines targeting critical functional domains of the protein.
Research with other ASFV proteins has shown that targeted gene deletion can produce attenuated strains that protect against challenge with virulent virus, as demonstrated with genes like P148R, I177L, and MGF360/505 gene families .
What methodological considerations are important when working with recombinant B117L protein?
When working with recombinant B117L protein, researchers should consider:
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Reconstitution protocol: Given its membrane protein nature, proper reconstitution is crucial. Manufacturers recommend dissolving lyophilized protein in distilled water to a concentration greater than 100 μg/ml .
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Storage conditions: To maintain protein stability, it's advised to store the protein at -20°C/-80°C, and aliquot reconstituted solutions to minimize freeze-thaw cycles .
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Functional assays: When assessing function, membrane-based assays are more relevant than solution-based assays due to B117L's natural membrane environment.
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Expression tag considerations: The presence of tags (like His-tags) should be considered when interpreting structural or functional data, as they may influence protein behavior.
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Membrane mimetics: For in vitro studies, appropriate membrane mimetics (detergents, nanodiscs, or liposomes) should be selected to maintain native-like structure and function.
How does B117L compare to other ASFV membrane proteins in functional studies?
While B117L functions in membrane permeabilization during ASFV infection, several other ASFV proteins also interact with cellular membranes with distinct functions:
| ASFV Protein | Primary Function | Subcellular Localization | Expression Timing |
|---|---|---|---|
| B117L | ER-derived envelope permeabilization | Membrane-associated | Early |
| E183L (p54) | Virus assembly, recruitment of viral membranes | Viral factories | Late |
| EP402R (CD2v) | Hemadsorption, host immune evasion | Cell surface | Late |
| p12 (O61R) | Membrane attachment | Viral envelope | Late |
| p17 (D117L) | Viral membrane formation | Precursor membranes | Late |
This comparative analysis highlights that B117L is somewhat unusual among ASFV membrane proteins for its early expression pattern and role in membrane permeabilization rather than structural roles in viral assembly .
What are the key challenges in studying B117L function?
Several challenges complicate B117L research:
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Membrane protein handling: As a membrane protein, B117L presents technical challenges for expression, purification, and functional characterization.
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Limited structural data: While computational predictions exist, high-resolution experimental structures remain limited.
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Undefined molecular mechanism: The precise molecular mechanism by which B117L permeabilizes membranes remains to be elucidated.
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Lack of specific inhibitors: Few specific inhibitors of B117L function have been identified, limiting pharmacological approaches to study its function.
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Cell culture limitations: ASFV primarily replicates in primary macrophages, which presents challenges for large-scale experiments compared to continuous cell lines.
What techniques can be used to investigate B117L interactions with host factors?
To identify and characterize potential B117L-host protein interactions, researchers can employ:
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Proximity labeling approaches: BioID or APEX2-based proximity labeling to identify proteins in close proximity to B117L during infection.
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Co-immunoprecipitation with mass spectrometry: To identify stable binding partners of B117L.
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Yeast two-hybrid screening: To identify potential protein-protein interactions in a membrane-based yeast two-hybrid system.
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CRISPR screens: To identify host genes whose disruption affects B117L function or ASFV replication.
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Comparative interactomics: Between B117L variants from different ASFV isolates to correlate interaction differences with functional outcomes.
How might understanding B117L function contribute to ASFV countermeasure development?
Understanding B117L function has several potential applications in developing ASFV countermeasures:
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Targeted antivirals: Small molecules that inhibit B117L membrane permeabilization activity could potentially block viral replication.
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Rational attenuation: Knowledge of B117L function could inform rational design of attenuated ASFV strains for vaccine development, similar to the approaches used with genes like I177L .
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Diagnostic markers: B117L or antibodies against it could serve as diagnostic markers for ASFV infection.
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Host-targeted approaches: Identification of essential host interactions could reveal host factors that could be targeted therapeutically.
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Cross-protective immunity: Understanding conserved functional domains of B117L across ASFV isolates could inform design of broadly protective vaccines.
