Recombinant African swine fever virus Uncharacterized protein B117L (Ken-095)

What is the B117L gene and what does it encode?

The B117L gene of ASFV encodes a 115-amino-acid integral membrane protein that is transcribed late during viral replication. This protein shows no homology to any previously characterized proteins . The B117L gene encodes a product that functions as a small membrane protein with viroporin-like activity, assisting in the permeabilization of the endoplasmic reticulum (ER)-derived envelope during ASFV infection . The protein represents approximately 0.16% of the virion protein mass, making it a minor component of the ASFV proteome .

What is the structural composition of the B117L protein?

The B117L protein consists of an N-terminal small globular ectodomain followed by a membrane domain (MD) spanning approximately 50 amino acids at the C-terminal end. The MD has a specific structure consisting of an amphipathic-strand–hydrophobic-helix–amphipathic-helix motif . Hydrophobicity distribution analysis confirms the presence of a single transmembrane helix (TMH) flanked by amphipathic sequences . Three-dimensional structure prediction further reveals this organization, with the main structural features being the N-terminal ectodomain and the C-terminal membrane domain .

Where does the B117L protein localize in cells during expression?

When expressed in mammalian cells (HEK293T and HeLa), the B117L protein preferentially localizes to the endoplasmic reticulum (ER). This was experimentally confirmed using confocal microscopy of cells transfected with B117L-GFP constructs, which showed robust colocalization with ER markers such as BiP-mCherry (a soluble ER marker) and mCherry-sec61β (a membrane ER marker) . The protein adopts a topology with its small globular ectodomain exposed to the lumen of the ER and its amphipathic-hydrophobic sequences inserted into the ER membrane . Subcellular localization predictions using DeepLoc 2.0 and LA(ProtT5) computational tools also supported B117L localization in the ER .

When is the B117L gene expressed during ASFV infection?

The B117L gene is transcribed late during the ASFV replication cycle. Time course experiments in primary swine macrophages infected with the ASFV strain Georgia (ASFV-G) showed that B117L RNA was first detected at 4 hours post-infection (hpi) and remained stable until 24 hpi . The expression kinetics of B117L were similar to that of the well-characterized late protein p72 (B646L), confirming its classification as a late gene . This late expression profile suggests the protein likely plays a role during viral assembly or entry rather than early replication events .

What evidence supports the viroporin-like function of B117L?

Multiple lines of evidence support the viroporin-like function of B117L:

• Membrane permeabilization activity: Peptides derived from the transmembrane helix (TMH) of B117L demonstrated the ability to permeabilize ER model membranes, particularly at low pH conditions that mimic the endosomal environment during viral entry .

• Ion channel formation: Experiments with partially overlapping peptides (PB117L-1, PB117L-2, and PB117L-3) showed that the TMH-containing peptide (PB117L-2) could establish pores and ion channels in membranes, particularly effective at low pH .

• Structural similarities to known viroporins: B117L shares structural similarities with the influenza A virus M2 protein, a well-characterized viroporin: both are single-pass, type III integral membrane proteins with an N-terminal ectodomain, a middle TMH, and a C-terminal amphipathic helix .

• Functional context: The protein's late expression profile and localization to the ER membrane are consistent with a role in viral entry and membrane permeabilization .

How do different ASFV isolates vary in their B117L gene, particularly regarding Ken05/Tk1?

Analysis of the B117L gene across different ASFV isolates revealed significant variation, particularly in the Ken05/Tk1 isolate. The Tajima's D value calculated specifically for the B117L gene present in the Ken05/Tk1 isolate was significantly different (P = 0.009 ≤ 0.05) compared to the rest of the isolates studied . This indicates that the B117L gene in Ken05/Tk1 may represent a significantly different phenotype .

BUSTED analysis identified amino acids 31, 32, 33, and 34, which belong to the ectodomain α-helix in most isolates, as potential sites associated with the phenotypic difference in Ken05/Tk1 . Three-dimensional structure prediction of the Ken05/Tk1 B117L protein showed that residues 31 to 34 could serve to cap a cognate structure at its C terminus, which is preserved but distorted in this isolate .

Interestingly, despite these differences in the ectodomain, no relevant sites under selection were predicted along the membrane domain (MD) of the protein in Ken05/Tk1, reinforcing the importance of this domain in performing a common function across all ASFV B117L proteins .

What experimental challenges exist in studying B117L gene function?

Several experimental challenges in studying B117L include:

• Gene deletion difficulties: Attempts to delete the B117L gene from the genome of the ASFV strain Georgia were unsuccessful in producing a pure recombinant virus lacking B117L. Only mixed virus populations were obtained, suggesting that B117L may be essential for virus growth .

• Functional complementation: The inability to isolate a pure B117L-deleted virus suggests that some critical functions of B117L could be complemented by the presence of B117L in other viral parental genomes, complicating functional studies .

• Protein expression systems: As an integral membrane protein that localizes to the ER, expression and purification of B117L for structural and functional studies present technical challenges typical for membrane proteins .

• Low abundance: B117L represents only about 0.16% of the virion protein mass, making detection and isolation from native sources difficult .

How does pH affect the activity of the B117L protein?

The activity of B117L, particularly its membrane-permeabilizing functions, is significantly enhanced at low pH. Experiments with giant unilamellar vesicles (GUVs) that mimicked the ER membrane composition showed that peptides representing the transmembrane domain of B117L (specifically peptide PB117L-2) had greater permeabilizing effects at pH 5.0 compared to neutral pH .

This pH-dependent activity is functionally relevant because it emulates conditions encountered during ASFV entry through the endocytic route, where endosomal acidification occurs . The enhancement of permeabilizing activity at low pH suggests that B117L functions may be triggered specifically during the endosomal stage of viral entry . This pH-dependent behavior is similar to other viral proteins involved in membrane fusion and permeabilization during entry, such as the influenza virus M2 protein .

What is the proposed role of B117L in the ASFV replication cycle?

Based on experimental evidence, the proposed role of B117L in the ASFV replication cycle is as follows:

• As a late gene product, B117L is likely incorporated into the ER-derived inner envelope during ASFV morphogenesis .

• During viral entry into new cells, as the virus passes through the endocytic pathway and encounters low pH environments, the membrane-permeabilizing activity of B117L is triggered .

• This activity helps in the permeabilization of the ER-derived viral envelope, facilitating the release of viral contents into the cytoplasm .

• The viroporin-like function of B117L thus assists in the uncoating process during viral entry, similar to the role of M2 protein in influenza virus infection .

The inability to isolate a pure B117L-deleted virus suggests this function is essential for ASFV replication .

What techniques can be used to study B117L membrane topology?

Several complementary techniques can be employed to study B117L membrane topology:

• Fluorescent protein fusion constructs: Using GFP fusion constructs with B117L and visualizing their localization in cells via confocal microscopy can provide insights into membrane orientation . In previous studies, various B117L constructs exhibited patterns consistent with ER localization and the formation of organized smooth ER (OSER) structures compatible with a single transmembrane helix having a cytoplasmic carboxy terminus .

• Immunofluorescence with epitope tags: Placing epitope tags at different positions (N-terminal, C-terminal, or internal loops) and using selective permeabilization protocols can help determine which portions of the protein are exposed to which cellular compartment .

• Protease protection assays: These can determine which domains of the protein are protected by the membrane and which are accessible to proteases, helping to establish the protein's orientation in the membrane .

• Glycosylation mapping: Introduction of artificial N-glycosylation sites at various positions can help determine which portions of the protein are exposed to the ER lumen, as glycosylation occurs only in the ER lumen .

How can the viroporin activity of B117L be experimentally assessed?

The viroporin activity of B117L can be assessed using the following methods:

• Membrane permeabilization assays: Using giant unilamellar vesicles (GUVs) that mimic the ER membrane composition, researchers can test the ability of B117L peptides to permeabilize membranes by monitoring the influx of fluorescent dyes (such as Alexa Fluor 488) into the vesicles .

• Ion channel activity measurements: Electrophysiological techniques such as patch-clamp recording or planar lipid bilayer experiments can directly measure ion channel formation and conductance properties of B117L .

• pH-dependent activity assessment: Testing membrane permeabilization and ion channel activities at different pH values (neutral versus acidic) can reveal how environmental pH affects B117L function, which is particularly relevant for understanding its role during viral entry through the endocytic pathway .

• Comparison with known viroporins: Using inhibitors of well-characterized viroporins (such as amantadine for influenza A M2) can help establish functional similarities or differences .

What evolutionary analysis methods are appropriate for studying B117L conservation?

Appropriate evolutionary analysis methods for studying B117L conservation include:

• Selective pressure analysis: Using algorithms like FUBAR (Fast Unconstrained Bayesian AppRoximation) to identify sites under negative or positive selection, which can reveal functionally important regions of the protein .

• Recombination detection: Using tools like GARD (Genetic Algorithm for Recombination Detection) to identify potential breakpoints in the gene sequence, which can indicate segments that have undergone recombination during evolution . Application of this method to B117L sequences revealed two potential break points at nucleotides 87 and 170 .

• Phylogenetic analysis: Constructing phylogenetic trees to visualize relationships between B117L sequences from different ASFV isolates and identify clusters or patterns of evolution .

• Structure-guided comparative analysis: Mapping conserved residues onto predicted or experimentally determined protein structures to identify functionally important regions .

• Statistical validation: Using statistical tests like Tajima's D to determine if sequence variations between isolates represent significantly different phenotypes .

How might understanding B117L function contribute to ASFV countermeasures?

Understanding B117L function has several potential applications for developing ASFV countermeasures:

• Antiviral drug targets: The viroporin-like activity of B117L makes it a potential target for antiviral drugs, similar to how amantadine targets the M2 protein of influenza A virus . Determining its molecular function opens up possibilities for developing specific inhibitors .

• Attenuated vaccine development: Knowledge of B117L's essential role in viral replication could inform the development of attenuated vaccine strains with modified but still functional B117L proteins .

• Entry inhibitors: As B117L appears to function during viral entry, compounds that specifically block its membrane-permeabilizing activity could prevent ASFV infection at an early stage .

• Structure-based drug design: Detailed structural information about B117L, particularly its transmembrane domain, could enable rational design of inhibitors that specifically interact with functionally important regions of the protein .

What techniques can be used to study B117L interactions with other viral or host proteins?

Several approaches can be employed to study potential interactions between B117L and other proteins:

• Co-immunoprecipitation (Co-IP): Using antibodies against B117L or tagged versions of the protein to pull down potential interaction partners, followed by mass spectrometry identification .

• Proximity labeling techniques: Methods such as BioID or APEX2, where B117L is fused to a promiscuous biotin ligase, can identify proteins in close proximity to B117L in living cells .

• Yeast two-hybrid screening: Although challenging for membrane proteins, modified versions of yeast two-hybrid systems can be used to screen for potential interaction partners .

• Fluorescence resonance energy transfer (FRET): Using fluorescently tagged B117L and potential interaction partners to detect close associations in live cells .

• Cross-linking mass spectrometry: Chemical cross-linking followed by mass spectrometry analysis can identify proteins that are physically associated with B117L .

Understanding these interactions could reveal additional roles for B117L in the viral life cycle beyond its viroporin-like activity.