Recombinant Acidianus bottle-shaped virus Putative transmembrane protein ORF85 (ORF85)

What is ORF85 and what are its basic structural features?

ORF85 is a putative transmembrane protein encoded by the Acidianus bottle-shaped virus (ABV), consisting of 85 amino acids with the sequence MVEIVGSNYFNFPPTTLIVLALGSAIAYKFLSNISTNPYVPAVLGIILVFLGHGGVISTIGAGITGLAISRAIGKDVFNFLSKVS . Structural analysis suggests it contains transmembrane domains with hydrophobic regions typical of membrane-associated proteins . The protein is part of ABV's 57 predicted open reading frames, most of which have unknown functions . While ORF85's precise role remains to be fully characterized, its predicted transmembrane nature suggests potential involvement in virus-host membrane interactions.

What is the genomic context of ORF85 within the Acidianus bottle-shaped virus?

ORF85 is encoded within the 23,814 bp linear double-stranded DNA genome of ABV, which has a G+C content of 35% and features a 590-bp inverted terminal repeat . The ABV genome contains 57 predicted ORFs, of which only three produce significant matches in public sequence databases: a glycosyltransferase, a thymidylate kinase, and a protein-primed DNA polymerase . ORF85 is not among these identifiable genes, highlighting its uniqueness. Notably, ABV shares only one homologous gene with other sequenced crenarchaeal viruses, emphasizing the evolutionary distinctiveness of this viral family .

What are the optimal expression systems for recombinant ORF85 production?

Multiple expression systems have been successfully used for ORF85 production, with Escherichia coli being the most commonly reported host . When expressed in E. coli, ORF85 is typically fused to an N-terminal histidine tag to facilitate purification . The following table summarizes reported expression systems for ORF85:

For optimal expression in E. coli, the methodology typically involves:

• Cloning the ORF85 sequence into an expression vector with an N-terminal His-tag

• Transformation into an E. coli expression strain (BL21(DE3) or similar)

• Induction with IPTG at reduced temperatures (16-25°C) to enhance solubility

• Cell lysis under native conditions with consideration for the protein's transmembrane nature

What purification strategies are most effective for recombinant ORF85?

Due to ORF85's transmembrane nature, purification requires special considerations. The most effective approach involves:

• Initial capture using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin, exploiting the N-terminal His-tag

• Buffer optimization with detergents to maintain solubility (typical buffers include Tris/PBS-based buffer with 6% Trehalose, pH 8.0)

• Additional purification steps may include ion exchange chromatography or size exclusion chromatography

• For long-term storage, lyophilization or storage in glycerol (up to 50%) at -20°C/-80°C is recommended

Purification yields protein with ≥90% purity as determined by SDS-PAGE . When working with ORF85, it's crucial to avoid repeated freeze-thaw cycles, with recommended storage of working aliquots at 4°C for up to one week .

What methods are most suitable for analyzing the structural characteristics of ORF85?

As a putative transmembrane protein, ORF85 presents challenges for structural characterization. Recommended approaches include:

• Membrane protein crystallography: While challenging, this could provide high-resolution structural data. The approach would require:

  • Large-scale protein production in a detergent-solubilized form

  • Screening of various detergents and lipids for crystallization

  • Use of lipidic cubic phase crystallization techniques

• Cryo-electron microscopy: Potentially more suitable than crystallography for membrane proteins, allowing visualization of ORF85 in a more native-like environment.

• Circular dichroism (CD) spectroscopy: For assessment of secondary structure content and thermal stability, particularly relevant given ABV's hyperthermophilic nature.

• Nuclear magnetic resonance (NMR) spectroscopy: For detailed structural analysis of specific domains, though the complete 85-amino acid protein might be challenging.

• Computational structure prediction: Given recent advances in AlphaFold and similar tools, computational prediction can provide valuable structural insights, especially when combined with experimental validation.

By analogy with structural studies on other archaeal viral proteins such as AFV1-99 (which was characterized up to 95°C) , special attention should be paid to ORF85's potential hyperthermostability when designing experimental conditions.

How can researchers investigate the membrane interaction properties of ORF85?

Given its putative transmembrane nature, several approaches can elucidate ORF85's membrane interactions:

• Liposome binding assays: Using fluorescently labeled ORF85 or liposomes to quantify binding affinity to membranes of various compositions.

• Planar lipid bilayer measurements: To investigate potential ion channel or pore-forming activity.

• Site-directed mutagenesis: Targeted mutations in the predicted transmembrane regions can help identify critical residues for membrane interactions.

• Membrane localization studies: Using fluorescently tagged ORF85 in archaeal host cells (if transfection systems are available) to verify subcellular localization.

• Lipid monolayer insertion experiments: To measure the ability of ORF85 to penetrate lipid interfaces.

When designing these experiments, the unique lipid composition of archaeal membranes should be considered, potentially incorporating archaeal-like lipids such as glycerol dialkyl glycerol tetraethers (GDGTs) that were identified in studies of other archaeal viruses .

What approaches can be used to identify potential interaction partners of ORF85?

Understanding protein-protein interactions is crucial for elucidating ORF85's function. Recommended methodologies include:

• Pull-down assays: Using His-tagged ORF85 as bait to capture interacting proteins from archaeal host lysates.

• Yeast two-hybrid screening: Though challenging for membrane proteins, modified split-ubiquitin systems can be employed.

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry can identify proximally located proteins in the viral particle or host membrane.

• Co-immunoprecipitation: Using antibodies against ORF85 to precipitate complexes for mass spectrometry analysis.

• Protein microarrays: Screening against arrays of archaeal host proteins to identify specific interactions.

When investigating potential DNA interactions, electrophoretic mobility shift assays (EMSA) can be employed, similar to those used for major coat proteins of Acidianus filamentous virus 1 .

How might ORF85 contribute to ABV's unique virion morphology and infection mechanism?

The bottle-shaped morphology of ABV is unique among known viruses, suggesting specialized structural proteins. To investigate ORF85's potential role:

• Cryo-electron tomography: To visualize ORF85's location within the ABV virion structure, potentially using gold-labeled antibodies against ORF85.

• Virus assembly assays: Investigating whether ORF85 is essential for correct virion assembly through genetic approaches or assembly inhibition.

• Host range determination: Testing whether ORF85 contributes to host specificity by comparing binding to different Acidianus strains.

• Time-course studies: Examining ORF85 expression and localization during different stages of infection.

Unlike other archaeal viruses like Sulfolobus spindle-shaped virus 1 (SSV1), where virion structural studies have been conducted , limited structural information exists for ABV, making these investigations particularly valuable.

What role might ORF85 play in ABV's adaptation to extreme environments?

ABV infects hyperthermophilic archaea that thrive at temperatures around 85°C and highly acidic pH (below 3) . To investigate ORF85's potential role in extremophilic adaptation:

• Thermal stability assays: Testing purified ORF85's stability at various temperatures (up to 95°C) and pH values (0-11), similar to studies performed on AFV1-99 .

• Structural analysis under extreme conditions: Using CD spectroscopy to monitor structural changes at varying temperatures and pH.

• Comparative genomics: Analyzing sequence conservation of ORF85 across viruses from different thermal environments.

• Adaptive laboratory evolution: Subjecting ABV to increasingly extreme conditions and monitoring genetic changes in ORF85.

Consider that ABV's adaptation to extreme conditions could involve specialized membrane interactions mediated by ORF85, which might be investigated through membrane fluidity measurements at varying temperatures.

How does ORF85 compare to transmembrane proteins from other archaeal viruses?

Comparative analysis can provide evolutionary and functional insights. Approaches include:

• Sequence-based comparisons: Using sensitive sequence search tools like HHpred to identify distant homologs in other viruses.

• Structural homology modeling: Based on any available structures of archaeal viral proteins, such as those from AFV1 or SIRV .

• Functional domain analysis: Identifying shared functional motifs that might suggest conserved mechanisms.

It's noteworthy that archaeal viruses exhibit remarkable morphological diversity and genetic uniqueness. For instance, rod-shaped archaeal viruses like ARV3 and MRV1 demonstrate biogeographic patterns in viral communities , suggesting environmental adaptations that might be reflected in membrane proteins like ORF85.

What can be learned from comparing ORF85 to the regions of ABV that show similarity to bacteriophage φ29 and human adenovirus?

ABV's genome shows surprising similarities to bacteriophage φ29 and human adenovirus in regions containing genes for protein-primed DNA polymerase and RNA structures similar to prohead RNA . To investigate potential evolutionary relationships:

• Phylogenetic analysis: Constructing phylogenetic trees of ORF85 and related proteins to trace evolutionary history.

• Functional complementation: Testing whether ORF85 can functionally replace homologous proteins in related viruses.

• Structural comparison: Determining if ORF85 shares structural features with proteins from these distantly related viruses.

These similarities support the concept of a primordial gene pool as a source of viral genes , and studying ORF85 may contribute to understanding viral evolution across domains of life.

How can ORF85 be used as a tool for studying archaeal virus-host interactions?

ORF85's potential involvement in host membrane interactions makes it valuable for studying infection mechanisms:

• Development of antibodies: Generating antibodies against ORF85 for immunolocalization studies during infection.

• Creation of fluorescently labeled ORF85: For real-time visualization of virus-host interactions.

• Design of inhibitory peptides: Based on ORF85 sequence to potentially block viral entry or assembly.

• Host receptor identification: Using ORF85 as bait to identify potential archaeal membrane receptors.

When designing experiments, consider methodologies used in studies of other archaeal viruses. For example, Acidianus tailed spindle virus (ATSV) researchers utilized a major coat protein cloning and expression approach with subsequent polyclonal antiserum production to study virus-host interactions .

What properties of ORF85 might be exploited for biotechnological applications?

Proteins from extremophilic organisms often possess unique properties valuable for biotechnology. For ORF85, consider:

• Thermostable protein scaffolds: If ORF85 demonstrates exceptional thermostability, it could serve as a scaffold for engineering thermostable enzymes.

• Membrane penetration technology: If ORF85 can penetrate membranes efficiently, it might be adapted for drug delivery systems.

• Archaeal expression systems: Knowledge gained from ORF85 could inform the development of expression systems for difficult-to-express membrane proteins.

• Structural biology tools: ORF85's potential stability under extreme conditions could make it useful for developing new approaches in structural biology.

Similar to how AFV1-99's remarkable stability (up to 95°C and pH 0-11) has made it interesting for biotechnological applications , ORF85's properties derived from adaptation to extreme environments could have unique applications.