Recombinant African swine fever virus Transmembrane protein B169L (War-086)

What is ASFV Transmembrane protein B169L (War-086)?

B169L (also known as War-086 in the Warthog/Namibia/Wart80/1980 isolate) is a structural protein of African swine fever virus (ASFV) with 169 amino acids. It belongs to the Asfarviridae family, which is endemic to sub-Saharan Africa and maintained through infection cycles between ticks and wild pigs, including bushpigs and warthogs . Despite being identified as a structural component of the virion, the specific function of B169L during viral replication remains unidentified . The protein exists in both full-length and truncated forms due to alternative splicing mechanisms during gene expression . ASFV causes a highly contagious hemorrhagic disease in domestic pigs, with clinical symptoms similar to classical swine fever, necessitating laboratory diagnosis for definitive identification .

What is the structure and topology of the B169L protein?

B169L possesses a distinctive hairpin transmembrane domain (HTMD) structure consisting of two transmembrane helices (TMHs) connected by a short linker sequence. Advanced bioinformatic analysis using DeepTMHMM demonstrates that these TMHs span approximately amino acids 29-49 and 62-80, with a connecting stretch between amino acids 50-61 . The protein adopts an Nout/Cout topology, where both the N-terminal and C-terminal regions face the cytoplasmic side of cellular membranes . Notably, B169L lacks a conventional signal peptide sequence, which is consistent with its classification as a type III membrane protein that directly inserts into the endoplasmic reticulum without signal peptide cleavage . This structural arrangement suggests that B169L may function as a viroporin, potentially forming membrane pores or channels during viral infection.

How is the B169L gene expressed in ASFV infection?

The B169L gene exhibits an early phase of transcription during ASFV infection, similar to the p30 gene (CP204L) but distinct from the late p72 gene (B646L). Quantitative PCR analysis reveals that B169L transcription is less affected by the DNA synthesis inhibitor AraC compared to p72, confirming its classification as an early gene .

The expression kinetics data for B169L compared to other ASFV genes is summarized in the following table:

This expression profile indicates that B169L is predominantly transcribed during the early phase of viral infection, suggesting its potential role in establishing initial infection or preparing the cellular environment for viral replication rather than virion assembly.

What is the cellular localization of B169L protein?

Confocal microscopy studies of cells transfected with B169L-GFP constructs demonstrate that the full-length B169L protein preferentially localizes to the endoplasmic reticulum (ER) . This localization was confirmed through co-transfection with specific markers for the ER, plasma membrane, and mitochondria, which consistently showed an ER-specific distribution pattern .

Interestingly, the truncated C-terminal B169L product (starting at Met92) and GFP-B169L fusion constructs with the fluorescent marker at the N-terminus both exhibited non-preferential distribution patterns similar to GFP alone . This observation suggests that proper membrane insertion and localization of B169L require the intact N-terminal region containing the first transmembrane helix, which likely functions as a 'Nexo' signal anchor to initiate membrane insertion . The specific ER localization of B169L implies its potential involvement in viral replication complex formation or modification of ER membrane permeability during infection.

How conserved is the B169L protein across different ASFV isolates?

Phylogenetic analysis of the B169L gene among representative ASFV isolates reveals a high degree of conservation, particularly in the regions encoding the transmembrane domains. Conservation plot analysis demonstrates scores around 9 (indicating high conservation) for residues 1-113, which encompasses the critical hairpin transmembrane domain . This conservation suggests strong evolutionary pressure to maintain the structural integrity of the transmembrane regions, highlighting their functional importance in the viral life cycle.

Beyond residue 113, there is decreased conservation with an area between residues 133-154 showing multiple deletions among distinct isolates, corresponding to variations in protein length among different phenotypes . Phylogenetic analysis of the complete B169L gene (~525 nucleotides) suggests the existence of at least three conserved phylogenetic groups, supported by nucleotide and amino acid differences revealed through pairwise analysis . Interestingly, the phylogenetic classification based on B169L does not consistently correlate with genotyping based on the p72 gene, suggesting that B169L evolved under different selective pressures .

What is the relationship between B169L and B438L genes in the ASFV genome?

The B169L and B438L genes exhibit a complex transcriptional relationship in the ASFV genome. These genes are arranged adjacently, with the B169L gene located upstream of the B438L gene . Through detailed transcriptional analysis, researchers have discovered that the distal promoter of the B438L gene, which is located within the B169L coding sequence, initiates the transcription of both the B438L mRNA and an alternatively spliced B169L mRNA (referred to as B169L mRNA2) .

This arrangement represents an intricate example of transcriptional economy in viral genomes, where a single promoter controls the expression of multiple genes. To map this relationship, researchers designed a series of promoter constructs to delineate the B438L promoter regions located within the B169L gene, using luciferase reporter assays to evaluate promoter activity . The B438L promoter region encompasses two distinct promoters, with the distal promoter initiating transcription of both B438L and the alternatively spliced B169L-2 . This shared transcriptional regulation suggests potential functional relationships between these proteins during the viral infection cycle.

How does the alternative splicing of B169L mRNA affect protein function?

The B169L gene produces two distinct mRNA species through alternative splicing: the full-length B169L mRNA encoding the complete 169-amino acid protein and the alternatively spliced B169L mRNA2 encoding a truncated protein (tpB169L) comprising amino acids 92-169 . This splicing event has significant structural and functional implications.

The full-length B169L protein contains the complete hairpin transmembrane domain, allowing it to properly insert into the ER membrane with both N- and C-termini facing the cytoplasm . In contrast, the truncated form lacks the N-terminal region and both transmembrane helices, resulting in a cytosolic protein with potentially distinct functions . Experimental evidence shows that while the full-length B169L-GFP localizes specifically to the ER, the truncated form exhibits non-preferential distribution throughout the cell, similar to GFP alone .

The transcription efficiency of B169L mRNA2 increases upon mutation of the initiation codon located upstream of the alternatively spliced B169L gene, suggesting complex regulation of protein isoform expression . This alternative splicing mechanism may represent a strategy for producing multiple protein isoforms with distinct subcellular localizations and functions from a single gene, expanding the functional repertoire of viral proteins during infection.

What experimental approaches can be used to study B169L's role in ASFV infection?

To elucidate the function of B169L in ASFV infection, researchers should employ a multidisciplinary approach combining molecular, cellular, and structural biology techniques:

• Gene Deletion and Mutational Analysis: Constructing B169L-deleted or mutated ASFV strains using the identified promoter regions is critical for assessing its role in viral replication and virulence . Site-directed mutagenesis of specific residues within the transmembrane domains can help determine structure-function relationships.

• Transcriptional Analysis: Quantitative PCR with specific primers and probes for B169L can be used to monitor gene expression kinetics during infection and compare with other viral genes . For B169L, researchers have successfully used primers (5'- TGAATGTAGATTTTATTGCGGGTATC-3' and 5'- AGGCCACAATGAAAGGA TTTTG-3') and probe (5′-FAM-AGGATGTTTTGAACGGTTCGCACG-MGB-NFQ-3′) designed based on the ASFV Georgia 2007/1 strain .

• Protein-Protein Interaction Studies: Co-immunoprecipitation, yeast two-hybrid screens, or proximity labeling techniques can identify viral and cellular proteins that interact with B169L, providing functional insights.

• Membrane Perturbation Assays: Given its potential viroporin-like activity, membrane permeabilization assays using liposomes or cell-based assays can assess how B169L affects membrane integrity and ion flux.

• Confocal Microscopy: Fluorescently tagged B169L constructs can be used to track its localization and dynamics during infection, as demonstrated by the successful localization of B169L-GFP to the ER .

• Phylogenetic Analysis: Comparative analysis of B169L sequences across ASFV isolates can reveal evolutionary conservation patterns indicative of functional constraints, as shown by the high conservation of the transmembrane domains .

How can recombinant B169L protein be effectively produced and purified for research?

Recombinant production of B169L presents challenges due to its transmembrane nature but can be achieved using several strategies:

• Expression System Selection: Escherichia coli has been successfully used for recombinant B169L production, as indicated by the commercial availability of E. coli-derived B169L (War-086) protein . For membrane proteins like B169L, specialized E. coli strains (C41(DE3), C43(DE3)) or eukaryotic systems like insect cells may improve folding and yield.

• Construct Design: Based on structural predictions, researchers should consider:

  • Full-length B169L (amino acids 1-169) for complete functional studies

  • The truncated form (amino acids 92-169) for comparative analysis

  • Isolated transmembrane domain constructs for biophysical studies

  • Fusion tags placed at the C-terminus to avoid interfering with membrane insertion, as N-terminal tags have been shown to disrupt proper localization

• Solubilization and Purification: Membrane proteins require careful extraction from cellular membranes using detergents. For B169L, mild non-ionic detergents like DDM (n-dodecyl-β-D-maltoside) or LMNG (lauryl maltose neopentyl glycol) are recommended initial choices for maintaining protein structure while solubilizing from membranes.

• Quality Control: Circular dichroism spectroscopy can verify the alpha-helical content expected from the transmembrane domains, while size exclusion chromatography can assess oligomeric state and homogeneity.

• Functional Validation: Liposome reconstitution assays can test if the purified protein retains membrane insertion capabilities and potential ion channel activities.

What are the potential functional implications of B169L's hairpin transmembrane domain?

The hairpin transmembrane domain (HTMD) of B169L, consisting of two transmembrane helices connected by a short linker, suggests several potential functional roles in ASFV infection:

• Viroporin Activity: The study titled "Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein" suggests that B169L may function as a viroporin . Viroporins are viral proteins that form pores in cellular membranes, potentially altering membrane permeability to ions or small molecules, which can facilitate viral entry, genome release, or virion assembly and release.

• Membrane Reorganization: The HTMD may participate in remodeling ER membranes to create viral replication factories, specialized compartments for viral genome replication and assembly.

• Protein Scaffolding: The conserved transmembrane regions might serve as anchoring domains that recruit other viral or cellular proteins to specific membrane sites, creating protein complexes necessary for viral replication.

• Host Cell Modulation: By inserting into ER membranes, B169L could potentially modulate host cellular responses such as the unfolded protein response or ER stress pathways, creating favorable conditions for viral replication.

• Structural Role in Virion: As a structural protein, the HTMD may contribute to the architecture of the viral envelope or the stability of the virion structure during morphogenesis.

The high conservation of the transmembrane regions across ASFV isolates underscores their functional importance and suggests strong evolutionary pressure to maintain these structural elements .

How does B169L expression timing relate to the ASFV replication cycle?

The temporal regulation of B169L expression provides insights into its potential functions during ASFV infection. Quantitative PCR analysis demonstrates that B169L exhibits early phase transcription kinetics, similar to the p30 gene but distinct from the late p72 gene .

When comparing gene expression (2^ΔΔCt) between infected cell cultures treated with the DNA synthesis inhibitor AraC versus mock-treated cells, B169L shows a difference of 1 × 10^1.94, comparable to p30 (1 × 10^1.82) but substantially lower than p72 (1 × 10^3.18) . This indicates significant transcriptional activity of B169L occurs before DNA replication, which is inhibited by AraC.

The early expression pattern suggests B169L likely functions in:

• Establishing favorable conditions for viral replication

• Modifying host cell membranes early in infection

• Participating in the formation of viral replication complexes

• Potentially counteracting early host defense mechanisms

This timing contrasts with late genes like p72, which are primarily involved in virion structural assembly. The coordinated expression of B169L with other early viral genes suggests it may function as part of protein complexes involved in preparing the cellular environment for efficient viral replication.

What methodologies are useful for studying B169L membrane insertion and topology?

Understanding the membrane insertion and topology of B169L requires specialized techniques adapted for transmembrane proteins:

• Protease Protection Assays: This approach can experimentally verify the predicted Nout/Cout topology by testing which protein regions are accessible to proteases in intact membrane preparations versus detergent-solubilized samples.

• Glycosylation Mapping: Introducing artificial glycosylation sites throughout the protein sequence can reveal which regions are exposed to the ER lumen (and thus glycosylated) versus cytoplasmic regions (non-glycosylated).

• Fluorescence Protease Protection (FPP): This technique combines fluorescent tags with selective permeabilization of cellular membranes and protease digestion to determine protein topology in living cells.

• Cysteine Accessibility Methods: Engineered cysteine residues throughout the protein can be tested for accessibility to membrane-impermeable sulfhydryl reagents, revealing their orientation relative to the membrane.

• Cryo-Electron Microscopy: For structural studies of B169L in membrane environments, cryo-EM of reconstituted protein in nanodiscs or liposomes can provide high-resolution insights into membrane insertion and oligomeric arrangements.

• Molecular Dynamics Simulations: Computational approaches can model how B169L's transmembrane helices insert into and stabilize within lipid bilayers, providing mechanistic insights that complement experimental data.

• Split-GFP Complementation: By fusing complementary GFP fragments to different termini of B169L, researchers can experimentally verify the predicted Nout/Cout topology based on fluorescence reconstitution patterns.

These methodologies would build upon the existing localization studies that have confirmed B169L's ER membrane insertion and provide deeper mechanistic insights into how this protein functions within cellular membranes during viral infection.