Recombinant African swine fever virus Uncharacterized protein B117L (Ba71V-083)
Description
Introduction
African swine fever virus (ASFV) represents a devastating pathogen responsible for a widespread pandemic affecting domestic and wild swine populations across Central Europe and East Asia, causing significant economic losses to the pork industry . The virus belongs to the Asfarviridae family and is endemic to sub-Saharan Africa, maintaining a natural cycle between ticks and wild pigs, bushpigs, and warthogs .
ASFV possesses a large double-stranded DNA genome containing more than 150 genes, many of which remained functionally uncharacterized until recently . Among these, the B117L gene has emerged as a subject of particular interest. The B117L gene corresponds to the Ba71V-083 locus in the Badajoz 1971 Vero-adapted strain (BA71V), which was the first ASFV genome to be completely sequenced . Recent research has demonstrated that B117L encodes a small membrane protein that plays a critical role in the permeabilization of the endoplasmic reticulum (ER)-derived envelope during ASFV infection .
Recombinant versions of the B117L protein have become valuable tools in ASFV research, facilitating structural studies, functional analyses, and development of potential countermeasures against ASFV infection. This article provides a comprehensive examination of recombinant ASFV B117L protein (Ba71V-083), detailing its molecular characteristics, functional properties, and applications in research and potential therapeutic interventions.
Gene Structure and Conservation
The B117L gene is present in the ASFV genome and encodes a protein of 115 amino acids that shows no homology to any previously published protein . The gene is transcribed late in the viral replication cycle, suggesting its involvement in the later stages of virion assembly or maturation . Evolutionary analysis has demonstrated high conservation of the transmembrane domain of B117L throughout ASFV evolution, indicating that the integrity of this domain is preserved by purifying selection . This conservation pattern underscores the functional importance of the transmembrane region in viral pathogenesis.
Research comparing the virulent BA71 strain with the attenuated BA71V strain has revealed that the B117L sequence is largely conserved between these variants, further supporting its essential role in viral biology . This high degree of conservation across different ASFV strains makes B117L a promising target for broad-spectrum antiviral interventions.
Protein Structure
B117L is a small integral membrane protein with a molecular weight of approximately 13.3 kDa in its native form . When expressed as a recombinant protein with tags (such as an N-terminal His tag), the molecular mass increases to about 25.65 kDa .
Structural analysis reveals that B117L contains a single transmembrane helix flanked by amphipathic sequences . This transmembrane helix, combined with the flanking regions, forms a membrane-associated C-terminal domain of approximately 50 amino acids . The secondary structure of B117L consists primarily of alpha-helices and beta-sheets stabilized by hydrogen bonds .
Hydrophobicity distribution analysis confirms the presence of the transmembrane region, which plays a crucial role in the protein's membrane interactions and ion channel formation capabilities . When expressed as a fusion protein with green fluorescent protein (GFP), B117L colocalizes with markers of the endoplasmic reticulum, indicating its preferential localization to this cellular compartment .
Physical and Biochemical Properties
Table 1: Key Physical and Biochemical Properties of Recombinant B117L
The B117L protein exhibits several notable physical and biochemical properties relevant to its function in viral infection. The transmembrane helix has the capacity to establish pores and ion channels in membranes, particularly at low pH . This property is characteristic of viroporins, a class of viral proteins that form pores in host cell membranes to facilitate various stages of the viral life cycle. Additionally, when expressed in cells, B117L can induce the formation of organized smooth ER (OSER) structures, consistent with its role in modifying cellular membranes during infection .
Membrane Interactions and Localization
B117L demonstrates strong affinity for cellular membranes, particularly the endoplasmic reticulum. When expressed as a GFP fusion protein in transient cell expression studies, B117L colocalizes with ER markers . This localization is consistent with its role in viral envelope formation and modification.
Intracellular localization studies of various B117L constructs have revealed a pattern indicative of the formation of organized smooth ER (OSER) structures . This observation aligns with the protein's structural characteristics, particularly its single transmembrane helix with a cytoplasmic carboxy terminus. The ability to induce OSER structures suggests that B117L plays an active role in remodeling cellular membranes, potentially to create specialized compartments for viral replication or assembly.
Viroporin-like Activity
One of the most significant discoveries regarding B117L is its viroporin-like properties. Experimental studies using partially overlapping peptides have demonstrated that the B117L transmembrane helix can establish pores and ion channels in membranes at low pH . This ability to permeabilize membranes is characteristic of viroporins, which play crucial roles in various stages of viral life cycles.
The viroporin-like function of B117L is consistent with its proposed role in assisting with the permeabilization of the ER-derived envelope during ASFV infection . This mechanism appears to be important for viral entry, potentially facilitating the release of viral contents into the host cell cytoplasm. The conservation of the transmembrane domain across ASFV strains further supports the critical nature of this function in viral replication and pathogenesis.
Role in Viral Replication Cycle
Table 2: Functional Roles of B117L in ASFV Infection
B117L is transcribed at late times during the ASFV replication cycle, suggesting its involvement in the later stages of viral assembly and maturation . Research indicates that the protein is essential for ASFV replication , underscoring its importance in the viral life cycle.
As a component of the viral envelope, B117L contributes to the structural integrity of the virion while facilitating interactions with host cell membranes during infection . Its ability to permeabilize membranes is particularly relevant during viral entry, when the virus must deliver its genetic material across cellular membranes to initiate replication.
Expression Systems and Purification
Recombinant B117L protein is typically produced using bacterial expression systems, with Escherichia coli being the predominant host organism . E. coli enables efficient production of the protein for research and development purposes, providing sufficient yields for structural and functional studies.
The recombinant protein is commonly expressed with affinity tags to facilitate purification and detection. The most frequently used modification is the addition of a histidine (His) tag, typically at the N-terminus of the protein . These tagged versions of B117L retain the functional properties of the native protein while allowing for simplified purification through affinity chromatography.
Commercial recombinant B117L preparations typically achieve purities exceeding 90% as determined by SDS-PAGE analysis . These preparations are available in various forms, including lyophilized powders or frozen liquids, and are typically formulated in phosphate-buffered saline (PBS) at pH 7.4 .
Vaccine Development
The strong humoral immune response induced by B117L makes it a potential target for vaccine development against ASFV . Recombinant B117L proteins could serve as subunit vaccine candidates or as components of multi-antigen vaccines designed to protect against ASFV infection.
Research on recombinant ASFV proteins, including B117L, is actively contributing to the development of effective vaccines against African swine fever . These efforts are critical given the devastating impact of ASFV on swine populations and the pork industry worldwide. The high conservation of B117L across ASFV strains further enhances its appeal as a vaccine antigen, potentially providing broad protection against various viral isolates.
Antiviral Drug Development
Table 4: Potential Therapeutic Applications of B117L Research
B117L's essential role in viral replication and its viroporin-like function make it an attractive target for antiviral drug development . Inhibitors targeting the B117L protein could potentially disrupt viral entry or replication, providing a novel approach to combating ASFV infection.
The well-conserved nature of B117L across different ASFV strains suggests that drugs targeting this protein might be effective against various isolates of the virus . This broad-spectrum potential enhances the appeal of B117L as a therapeutic target in the ongoing effort to control ASFV outbreaks globally.
Host-Pathogen Interaction Studies
Investigation of B117L's interactions with host cell proteins could reveal additional aspects of its role in viral pathogenesis. Identifying host factors that interact with B117L might uncover new targets for therapeutic intervention or enhance our understanding of ASFV's strategies for modulating host cell functions.
Improved Recombinant Production Systems
Development of optimized expression systems for recombinant B117L could enhance protein yield and quality for research and potential therapeutic applications. Alternative expression hosts, such as mammalian or insect cells, might produce recombinant B117L with post-translational modifications more closely resembling the native viral protein.
Combinatorial Approaches for Vaccine Development
Exploring combinations of B117L with other ASFV antigens in vaccine formulations might provide enhanced protection against viral infection. Multivalent vaccines targeting multiple viral components could reduce the risk of immune escape through mutation of individual antigens.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
Note: While the tag type is determined during production, we prioritize fulfilling requests for specified tags if provided in advance.
Target Background
KEGG: vg:22220313
Q&A
What is the basic structure of the ASFV B117L protein?
B117L is a 115-amino-acid integral membrane protein transcribed during the late stages of the ASFV replication cycle. Hydrophobicity distribution analysis confirms the presence of a single transmembrane helix (TMH), which in combination with flanking amphipathic sequences, forms a membrane-associated C-terminal domain of approximately 50 amino acids .
3D-structure prediction using AlphaFold reveals a single helix structure (residues 67 to 114) that includes the transmembrane helix moiety. Upon insertion into a phospholipid bilayer, this long helix is preceded at the N-terminus by a solvent-exposed small globular ectodomain containing a defined, shorter helix. The membrane-spanning helix extends into the cytosol, exposing its C-terminal stretch (residues 90 to 114) to solvent .
Table 1: Structural domains of B117L protein
| Domain | Residue Range | Characteristics |
|---|---|---|
| N-terminal domain | 1-66 | Solvent-exposed, includes small globular ectodomain |
| Transmembrane helix | 67-89 | Membrane-spanning region |
| C-terminal stretch | 90-114 | Extends into cytosol, solvent-exposed |
How is B117L localized within infected cells?
Methodological approach: To determine the subcellular localization of B117L, researchers performed ectopic transient cell expression of the B117L gene as a green fluorescent protein (GFP) fusion protein. The expressed protein was visualized using fluorescence microscopy and compared with known cellular markers.
Results demonstrate that B117L colocalizes with markers of the endoplasmic reticulum (ER). Intracellular localization of various B117L constructs displays a pattern consistent with the formation of organized smooth ER (OSER) structures. This localization pattern is compatible with the presence of a single transmembrane helix with a cytoplasmic carboxy terminus .
What is the predicted molecular weight of recombinant B117L protein?
The recombinant B117L protein expressed in E. coli has a predicted molecular weight of 25.7 kDa . This is higher than the weight of the native protein due to the addition of tags and fusion partners used in recombinant expression systems. When expressed as a GFP fusion protein for localization studies, the molecular weight increases further to incorporate the approximately 27 kDa contributed by GFP.
What expression systems are optimal for producing recombinant B117L protein?
E. coli expression systems have been successfully used to produce recombinant B117L protein. The commercially available recombinant protein is expressed in E. coli with an N-terminal His-tag, resulting in a predicted molecular weight of 25.7 kDa with purity >90% as verified by SDS-PAGE .
For experimental studies, researchers should consider:
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Codon optimization for E. coli expression
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Selection of appropriate tags (His, GST) for purification
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Optimization of induction conditions to maximize protein yield
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Inclusion of protease inhibitors during purification
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Verification of proper folding using circular dichroism spectroscopy
Given that B117L is a membrane protein, researchers might encounter solubility issues. Alternative expression systems to consider include:
Table 2: Comparison of expression systems for recombinant membrane proteins
| Expression System | Advantages | Disadvantages | Suitability for B117L |
|---|---|---|---|
| E. coli | Fast, inexpensive, high yield | May form inclusion bodies | Good for initial studies |
| Insect cells | Better folding of eukaryotic proteins | More expensive, longer production time | Better for functional studies |
| Mammalian cells | Native-like post-translational modifications | Expensive, lower yield | Best for interaction studies |
What methodologies are effective for studying B117L's ion channel activity?
Experimental approach for ion channel activity studies:
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Peptide synthesis: Generate partially overlapping peptides corresponding to the transmembrane helix region of B117L.
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Membrane interaction assays: Assess the capacity of these peptides to establish pores and ion channels in membranes at varying pH conditions.
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Electrophysiology measurements: Perform voltage clamp experiments using lipid bilayers containing reconstituted B117L protein or peptides.
Research findings demonstrate that the B117L transmembrane helix has the capacity to establish pores and ion channels in membranes, particularly at low pH conditions . This suggests a viroporin-like function, which may be essential for permeabilizing cellular membranes during viral entry or egress.
How can researchers effectively study the role of B117L in ASFV replication?
To study B117L's role in viral replication, researchers should implement a multifaceted approach:
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Gene knockout studies: Generate B117L deletion mutants using CRISPR-Cas9 or similar genome editing techniques as demonstrated for other ASFV genes .
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Complementation assays: Rescue viral growth defects by providing B117L in trans to confirm specificity.
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Time-of-addition experiments: Add B117L-specific inhibitors at different time points post-infection to identify the stage at which B117L functions.
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Co-immunoprecipitation: Identify interaction partners of B117L during viral infection.
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High-resolution microscopy: Track B117L distribution during different stages of viral replication.
Recent studies support a viroporin-like assistant role for the B117L gene-encoded product in ASFV entry , suggesting that experiments focusing on early infection events may be most informative.
How conserved is the B117L protein across different ASFV isolates?
Methodological approach for evolutionary analysis:
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Sequence alignment of B117L genes from diverse ASFV isolates
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Calculation of synonymous vs. non-synonymous substitution rates (dN/dS)
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Analysis of site-specific selection pressures
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Phylogenetic reconstruction
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Statistical testing for significant phenotypic differences
This conservation pattern suggests that B117L performs an essential function in the viral life cycle, with particular importance attributed to the transmembrane domain.
What methods can detect specific B117L phenotypes evolving under positive selection?
To identify B117L phenotypes evolving under positive selection, researchers should implement:
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Maximum likelihood methods: Apply codon-based models that allow for variable selection pressures across sites.
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Branch-site models: Test for positive selection along specific lineages.
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Ancestral sequence reconstruction: Infer historical amino acid changes.
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Structural mapping of variable sites: Identify regions under different selection pressures.
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Functional verification: Test phenotypic differences experimentally.
Research findings indicate that the B117L gene in the isolate Ken05/Tk1 may represent a significantly different phenotype compared to other isolates (P = a significantly different phenotype compared to other isolates (P = 0.009 ≤ 0.05), warranting further investigation into its functional implications .
How does B117L contribute to ASFV entry and infection?
B117L appears to function as a viroporin-like protein that assists in the permeabilization of the ER-derived envelope during ASFV infection . This function is likely critical during viral entry.
The methodological framework for studying this function includes:
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Viral entry assays: Quantify entry efficiency in the presence of B117L inhibitors or antibodies.
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Membrane permeabilization assays: Measure the ability of B117L to permeabilize membranes using fluorescent dyes.
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pH-dependent activity studies: Assess how endosomal acidification affects B117L function.
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Electron microscopy: Visualize membrane deformation and pore formation.
Research findings indicate that the transmembrane helix of B117L can establish spores and ion channels in membranes at low pH , suggesting that it may facilitate viral genome release during entry by permeabilizing endosomal membranes.
What is the relationship between B117L and endoplasmic reticulum stress during ASFV infection?
ASFV infection induces ER stress, and B117L appears to be involved in modulating ER functions . The research approach to explore this relationship should include:
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ER stress marker analysis: Measure activation of key ER stress indicators (BiP, CHOP, XBP1 splicing) in the presence and absence of B117L expression.
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Unfolded protein response (UPR) pathway assessment: Evaluate activation of PERK, IRE1, and ATF6 branches of the UPR.
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Calcium homeostasis studies: Measure cytoplasmic calcium levels, as B117L may affect this through its ion channel activity.
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Inhibitor studies: Test how pharmacological inhibition of ER stress pathways affects B117L localization and function.
Preliminary findings suggest that differentially expressed genes associated with UPR are significantly enriched upon ASFV infection, and B117L may play a role in this process . The relationship between B117L and activating transcription factor 6 (ATF6), a key component of the UPR, warrants further investigation.
How can B117L be utilized in ASFV vaccine development strategies?
B117L's essential role in viral entry makes it a potential target for vaccine development. A systematic approach would include:
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Immunogenicity assessment: Determine if B117L elicits neutralizing antibodies or cellular immunity in immunized animals.
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Epitope mapping: Identify immunogenic regions of B117L, particularly those accessible on the virion surface.
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Attenuated virus development: Create ASFV variants with modified B117L that replicate poorly but maintain immunogenicity.
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Subunit vaccine formulation: Incorporate B117L or its immunogenic fragments into subunit vaccines.
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Protection studies: Evaluate if B117L-based immunization protects against challenge with virulent ASFV.
While there are currently no reports of B117L being used specifically in vaccine studies, its conservation and essential function make it a promising candidate. Other ASFV proteins have been tested in vaccine trials with partial success , suggesting a multi-antigen approach including B117L might be effective.
What are the optimal methods for detecting B117L protein in diagnostic applications?
For developing B117L-based diagnostics, researchers should consider:
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Antibody development: Generate monoclonal antibodies against conserved epitopes of B117L.
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ELISA optimization: Develop assays using recombinant B117L as an antigen to detect anti-B117L antibodies in infected animals.
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Multiplexed approaches: Combine B117L with other ASFV antigens for improved sensitivity.
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PCR-based detection: Design primers specific to the B117L gene for molecular diagnosis.
Existing ASFV diagnostic methods utilize several viral proteins such as p30, p54, p72, and pB602L in recombinant protein-based ELISAs . Including B117L in such panels could potentially enhance sensitivity and specificity, particularly for detecting antibodies against diverse ASFV isolates.
How can synthetic genomics approaches be applied to study B117L function?
Recent advances in synthetic genomics offer powerful tools for studying ASFV genes like B117L:
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Genome assembly: TAR (transformation-associated recombination) assembly techniques can be used to construct artificial ASFV genomes with modified B117L genes .
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Gene replacement: Replace native B117L with tagged or mutated versions to study functional domains.
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Reporter gene insertion: Insert fluorescent reporters adjacent to B117L to monitor expression kinetics.
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Combinatorial genome engineering: Create libraries of B117L variants to screen for functional changes.
A synthetic genomics approach has been successfully applied to ASFV, allowing for engineering modifications including gene deletions and fluorescent marker insertions . This technology could be leveraged to create B117L variants for comprehensive functional analysis.
What transcriptomic approaches reveal insights about B117L expression during infection?
To understand B117L expression patterns:
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CAGE-seq analysis: Map the transcription start sites of B117L with single-nucleotide resolution.
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RNA-seq time course: Profile B117L expression at different stages of infection.
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Single-cell transcriptomics: Identify cell-to-cell variation in B117L expression.
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Promoter characterization: Define the regulatory elements controlling B117L expression.
Studies have shown that B117L is transcribed late during the virus replication cycle , suggesting it may function primarily in virion assembly or egress. Applying CAGE-seq and other techniques used for ASFV transcriptome analysis would provide detailed insights into the regulation of B117L expression.
