Recombinant Acidianus bottle-shaped virus Putative transmembrane protein ORF91a (ORF91a)

What is ORF91a and what organism does it come from?

ORF91a is a putative transmembrane protein encoded by the Acidianus bottle-shaped virus (ABV). This virus infects Acidianus species, which are hyperthermophilic archaea that thrive in acidic hot springs with temperatures exceeding 85°C and pH below 3, such as those found in Yellowstone National Park . The protein is 91 amino acids in length and contains predicted transmembrane domains, suggesting it may play a role in viral-host membrane interactions during infection .

Acidianus species belong to the order Sulfolobales within the phylum Crenarchaeota . These extremophiles have adapted to survive in harsh environments, including acidic hot springs and hydrothermal vents. The viruses that infect these organisms, including ABV, have evolved specialized proteins like ORF91a that function under extreme conditions, making them interesting subjects for both basic and applied research.

What are the basic structural characteristics of ORF91a?

ORF91a exhibits several key structural features consistent with a transmembrane protein:

• Protein length: 91 amino acids

• Transmembrane domains: Contains hydrophobic regions consistent with membrane-spanning segments

• Tag compatibility: Can be expressed with an N-terminal His-tag without disrupting protein structure

• Predicted topology: Likely contains multiple transmembrane segments based on its amino acid composition

The hydrophobicity profile of ORF91a reveals distinct hydrophobic regions (amino acids 18-40 and 55-77) that likely form transmembrane helices. These regions are characterized by stretches of predominantly hydrophobic amino acids, including valine, isoleucine, leucine, and alanine, which are typical components of transmembrane domains. The protein also contains charged and polar residues at the predicted cytoplasmic and extracellular regions, consistent with the positive-inside rule for membrane protein topology.

What is known about the function of ORF91a?

The exact function of ORF91a remains uncharacterized, but based on its features and context, several hypotheses can be proposed:

• Membrane modification: ORF91a may alter host cell membranes during infection

• Viral assembly: It could participate in virion assembly or viral release

• Host interaction: May mediate specific interactions with host proteins

• Structural role: Could form part of the viral envelope or capsid structure

By analogy with other archaeal viral transmembrane proteins, ORF91a might function in a manner similar to proteins from related viruses. For example, in Sulfolobus islandicus rod-shaped virus 2 (SIRV2), transmembrane proteins are involved in viral egress through formation of pyramidal structures . While no specific pathway or protein interactions have been definitively identified for ORF91a in the available data , its transmembrane nature strongly suggests a role in viral-host membrane interactions.

How is Acidianus bottle-shaped virus classified taxonomically?

Acidianus bottle-shaped virus (ABV) belongs to a diverse group of archaeal viruses that infect extremophilic archaea. The taxonomic classification of ABV is:

ABV has a distinctive bottle-shaped morphology that differentiates it from other archaeal viruses like the filamentous Lipothrixviridae (e.g., Acidianus filamentous virus 1) or the rod-shaped Rudiviridae. The morphological and genetic diversity of archaeal viruses is extraordinarily high, with viruses displaying unique structures not seen in bacterial or eukaryotic viruses .

What experimental approaches can be used to determine the membrane topology of ORF91a?

Multiple complementary approaches should be employed to confidently determine ORF91a's membrane topology:

• Cysteine scanning mutagenesis and accessibility analysis:

  • Systematically replace native residues with cysteine throughout the protein

  • Treat with membrane-impermeable sulfhydryl reagents

  • Residues accessible to reagents are likely exposed to the aqueous environment

  • This approach can be performed in native archaeal hosts or reconstituted systems

• Fusion protein reporters:

  • Create fusion constructs with reporter domains (e.g., GFP, alkaline phosphatase)

  • Position reporters at different locations in the protein sequence

  • Analyze reporter activity/fluorescence to determine orientation

  • Consider using thermostable reporter variants due to extremophilic origin

• Protease protection assays:

  • Express ORF91a in membrane vesicles

  • Treat with proteases under varying conditions of membrane permeabilization

  • Map protected fragments by mass spectrometry

  • This approach requires careful control of experimental conditions due to the thermophilic nature of the protein

• Computational prediction validation:

  • Compare experimental results with predictions from multiple topology prediction algorithms

  • Reconcile discrepancies between methods

  • Use structural models to interpret experimental data

When working with archaeal proteins, consider the adaptation to extreme conditions. Experimental procedures may need modification to account for the protein's stability at high temperatures and low pH.

How can the interaction partners of ORF91a be identified in the viral life cycle?

Identifying interaction partners of ORF91a requires strategies adapted for extremophilic proteins:

• Co-immunoprecipitation under native conditions:

  • Generate antibodies against recombinant ORF91a

  • Perform pull-downs from infected Acidianus cells

  • Identify co-precipitating proteins by mass spectrometry

  • Maintain buffers at appropriate pH and temperature to preserve native interactions

• Proximity labeling approaches:

  • Genetically fuse ORF91a with enzymes like BioID or APEX2

  • Express in host cells and activate labeling during infection

  • Identify biotinylated proteins using streptavidin purification and mass spectrometry

  • Adapt protocols for high temperature and low pH conditions

• Yeast two-hybrid with specialized libraries:

  • Create specialized libraries from Acidianus genomic DNA and ABV viral DNA

  • Use thermotolerant yeast strains if possible

  • Screen for interactions with ORF91a as bait

  • Verify positive interactions with secondary assays

• Cross-linking mass spectrometry:

  • Treat infected cells with membrane-permeable crosslinkers

  • Isolate ORF91a and identify crosslinked peptides by MS

  • This technique can capture transient interactions

  • Requires optimization for extremophilic conditions

• Split-reporter complementation assays:

  • Create fusion proteins with split fluorescent protein halves

  • Test candidate interactions in heterologous systems

  • Signal indicates protein proximity in living cells

Based on studies of related archaeal viruses, potential interaction partners may include other viral structural proteins, host membrane proteins, or components involved in viral assembly and egress pathways .

What structural biology techniques are most suitable for studying ORF91a?

Several structural biology techniques can be applied to ORF91a, each with specific advantages:

• X-ray crystallography:

  • Requires purification of milligram quantities of stable, homogeneous protein

  • May need to use lipidic cubic phase for membrane protein crystallization

  • Can provide atomic-resolution structures

  • Challenge: obtaining well-diffracting crystals of membrane proteins

• Cryo-electron microscopy (cryo-EM):

  • Single-particle analysis for larger complexes containing ORF91a

  • Suitable for membrane proteins in native-like environments

  • Can visualize different conformational states

  • Challenge: small size of ORF91a may require fusion to larger scaffolds

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Suitable for small membrane proteins (<30 kDa)

  • Can provide dynamic information in addition to structure

  • Requires isotopic labeling (15N, 13C, 2H)

  • Challenge: maintaining protein stability in detergent micelles

• Solid-state NMR:

  • Can study ORF91a in lipid bilayers

  • Provides orientation information of transmembrane helices

  • May be more representative of native environment

  • Challenge: requires specialized equipment and expertise

• Integrated structural approaches:

  • Combine multiple techniques (SAXS, EPR, FRET, HDX-MS)

  • Each method provides complementary information

  • Build comprehensive structural models

Previous studies with archaeal viral proteins have successfully used X-ray crystallography to determine structures, as demonstrated with AFV1-157, which revealed a novel fold with nuclease activity . For ORF91a, detergent screening and lipid reconstitution will be critical steps for any structural study.

How does temperature and pH affect the stability and function of ORF91a?

Given that ORF91a comes from a virus that infects extremophilic archaea, its stability and function are likely optimized for high temperature and low pH conditions:

• Temperature effects:

  • Thermal stability likely exceeds that of mesophilic proteins

  • Optimal activity probably occurs at temperatures >70°C

  • Contains adaptations for thermostability:

    • Increased hydrophobic core packing

    • Higher proportion of charged residues forming salt bridges

    • Reduced loop regions susceptible to denaturation

• pH dependency:

  • Likely shows optimal stability and activity at acidic pH (pH 2-4)

  • May contain increased proportion of acidic residues on surface

  • Protonation state of key residues may regulate function

• Experimental approaches to characterize stability:

  • Circular dichroism spectroscopy at varying temperatures and pH

  • Differential scanning calorimetry to determine melting temperatures

  • Activity assays across pH and temperature ranges

  • Protein unfolding studies with chemical denaturants

• Methodological considerations:

  • Design experiments to mimic native conditions (>85°C, pH <3)

  • Use thermostable buffers that maintain pH at high temperatures

  • Consider implementing pressure systems for super-heated reactions

For recombinant expression, the protein may need to be refolded under conditions that mimic its native environment, as E. coli expression systems operate at much lower temperatures and neutral pH .

What methodologies can be used to study ORF91a's role during viral infection?

Studying ORF91a's role during viral infection requires specialized approaches for extremophilic systems:

• Genetic manipulation of the viral genome:

  • Generate ORF91a deletion mutants or point mutations

  • Create tagged versions for localization studies

  • Complementation studies to verify phenotypes

  • Challenge: limited genetic tools for archaeal viral systems

• Time-course infection experiments:

  • Monitor expression of ORF91a at different stages using RT-qPCR

  • Compare with data from related archaeal viruses, where genes show temporal expression patterns

  • Correlate with morphological changes in host cells

• Localization studies:

  • Generate antibodies against ORF91a for immunofluorescence

  • Use fluorescently tagged versions if genetically tractable

  • Perform subcellular fractionation followed by Western blotting

  • Electron microscopy with immunogold labeling

• Functional inhibition approaches:

  • Develop peptide inhibitors targeting predicted functional domains

  • Apply during different stages of infection

  • Measure impacts on viral replication and assembly

• Transcriptomic and proteomic profiling:

  • Compare wild-type infection with ORF91a mutant infections

  • Identify pathways affected by ORF91a disruption

  • Similar to approaches used with SIRV2 infection studies

When designing these experiments, consider the temporal expression patterns observed in other archaeal viruses. For example, in SIRV2, structural proteins like the major coat protein (ORF134) and viral assembly protein (ORF98) show increased expression late in infection, suggesting different functional roles at different stages .

How can transcriptomic data be analyzed to understand ORF91a expression during viral infection?

Analyzing transcriptomic data for ORF91a expression requires specialized approaches for archaeal systems:

• Time-course RNA-seq experimental design:

  • Sample at multiple timepoints post-infection (e.g., 0, 1, 2, 3, 5, 8, 12 hours)

  • Include mock-infected controls

  • Perform biological replicates (minimum n=3)

  • Use RNA extraction methods optimized for extremophiles

• Data normalization strategies:

  • Consider specialized normalization for viral transcripts

  • Use spike-in controls for absolute quantification

  • Calculate RPKM/FPKM/TPM values for comparison across samples

  • Compare methods used in similar studies of archaeal viruses

• Expression pattern classification:

  • Group genes by expression patterns (early, middle, late)

  • Compare ORF91a expression with genes of known function

  • Create expression clusters to identify functionally related genes

• Co-expression network analysis:

  • Build co-expression networks to identify genes with similar patterns

  • Use weighted gene correlation network analysis (WGCNA)

  • Identify modules containing ORF91a

  • Compare with expression data from other archaeal viruses

• Integration with genomic location:

  • Analyze expression in context of genomic location

  • Map reads to the virus genome to identify transcription start sites

  • Identify potential polycistronic messengers

Based on studies of SIRV2, viral gene expression often shows distinct temporal patterns. Some genes reach peak expression early (1-2 hours post-infection), while structural genes tend to increase throughout infection . Analyzing whether ORF91a follows early or late expression patterns can provide clues about its function.

What bioinformatic approaches can help predict the function of ORF91a?

Multiple bioinformatic approaches can be integrated to predict ORF91a function:

• Sequence-based analyses:

  • PSI-BLAST against diverse databases to find remote homologs

  • HHpred for sensitive profile-profile comparisons

  • Analysis of conserved domains and motifs

  • Search for sequence signatures of known functional families

• Structural prediction and analysis:

  • Use AlphaFold2 or RoseTTAFold for structure prediction

  • Structural alignment against known protein structures

  • Identification of potential binding pockets or active sites

  • Electrostatic surface analysis for interaction interfaces

• Evolutionary analyses:

  • Identify orthologs in related archaeal viruses

  • Perform comparative genomic analyses across viral families

  • Calculate evolutionary rates to identify conserved regions

  • Synteny analysis to identify genomic context conservation

• Functional annotation transfer:

  • Integrate evidence from multiple sources (sequence, structure, context)

  • Use tools like COFACTOR for structure-based function prediction

  • Employ Gene Ontology term prediction algorithms

  • Consider specialized viral protein databases

• Network-based approaches:

  • Predict protein-protein interactions using interolog mapping

  • Analyze genomic neighborhood for functional associations

  • Use guilt-by-association methods with co-expressed genes

For ORF91a specifically, transmembrane topology prediction tools (TMHMM, Phobius) should be applied to identify membrane-spanning regions, which will inform functional hypotheses about its role in membrane-associated processes.

How should contradictory experimental results about ORF91a's function be reconciled?

When faced with contradictory results about ORF91a function, a systematic reconciliation approach is essential:

• Critical assessment of experimental conditions:

  • Compare temperature, pH, and buffer conditions between studies

  • Assess protein preparation methods (tags, purification approaches)

  • Evaluate expression systems (E. coli vs. archaeal hosts)

  • Consider whether native conditions were adequately replicated

• Orthogonal validation experiments:

  • Design experiments using different methodologies

  • Test function under varied conditions mimicking infection stages

  • Employ both in vitro and in vivo approaches

  • Use native host systems when possible

• Contextual interpretation framework:

  • Consider that ORF91a may have multiple distinct functions

  • Different domains may perform separate roles

  • Function may be condition-dependent or state-dependent

  • Context (viral infection stage, host interaction) may alter function

• Integrative data analysis:

  • Weight evidence based on methodological rigor

  • Create testable models that accommodate disparate results

  • Apply Bayesian approaches to update functional hypotheses

  • Meta-analysis of all available data

• Collaborative resolution strategies:

  • Organize inter-laboratory validation studies

  • Share reagents and protocols to ensure reproducibility

  • Establish community standards for archaeal virus research

Given the limited information available specifically about ORF91a, any contradictory results should be interpreted in the context of what is known about other archaeal viral proteins, while acknowledging the unique biological context of hyperthermophilic archaeal systems.

What statistical methods are appropriate for analyzing ORF91a protein-protein interaction data?

Analyzing protein-protein interaction data for ORF91a requires specialized statistical approaches:

• Filtering and scoring interaction data:

  • Implement appropriate scoring algorithms for different detection methods:

    • MS-based: SAINT, CompPASS, or MIST for spectral counting

    • Y2H: Statistical filtering based on growth phenotypes

    • Split-reporter systems: Signal-to-noise thresholding

  • Use appropriate negative controls for background estimation

  • Account for protein abundance in calculating interaction probabilities

• Network analysis approaches:

  • Calculate network centrality measures to identify key interactions

  • Perform clustering analysis to identify functional modules

  • Apply Markov clustering or other community detection algorithms

  • Calculate betweenness centrality to identify bridging interactions

• Statistical validation methods:

  • Permutation tests to establish significance of network features

  • Bootstrap sampling to establish confidence intervals

  • Hypergeometric tests for enrichment of functional categories

  • False discovery rate control for multiple testing correction

• Integration with existing knowledge:

  • Bayesian integration of new data with prior information

  • Calculate likelihood ratios for candidate interactions

  • Assess consistency with known viral protein interaction networks

  • Compare with interaction networks from related viruses

• Visualization and interpretation tools:

  • Use tools like Cytoscape for network visualization

  • Implement GO term enrichment analysis for interacting partners

  • Apply edge bundling for complex networks

  • Develop dynamic models of interaction changes during infection

When analyzing interaction data for archaeal viral proteins like ORF91a, special consideration should be given to the unique biological context. Standard interaction databases may have limited coverage of archaeal systems, requiring customized reference sets.

What are the optimal conditions for expressing recombinant ORF91a in E. coli?

Expressing archaeal viral proteins like ORF91a in E. coli requires optimization strategies:

• Expression system selection:

  • pET vector systems with T7 promoter for high expression

  • Consider low-copy vectors for toxic membrane proteins

  • Test multiple E. coli strains (BL21(DE3), C41/C43, Rosetta for rare codons)

  • Evaluate expression with different fusion tags (His, MBP, SUMO)

• Induction conditions optimization:

  • Test range of IPTG concentrations (0.1-1.0 mM)

  • Evaluate different induction temperatures (16°C, 25°C, 30°C)

  • Consider extended expression times at lower temperatures

  • Test auto-induction media for gradual protein production

• Optimizing solubility and folding:

  • Co-express with molecular chaperones (GroEL/ES, DnaK)

  • Include mild detergents in lysis buffer for membrane proteins

  • Test various solubilization conditions

  • Consider fusion to solubility-enhancing partners

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Size exclusion chromatography for final polishing

  • Buffer optimization (pH 7-8, 150-300 mM NaCl, glycerol 5-10%)

  • Include stabilizing agents if needed

• Quality control assessments:

  • SDS-PAGE for purity evaluation (>90% purity achievable)

  • Western blotting for identity confirmation

  • Mass spectrometry for molecular weight verification

  • Dynamic light scattering for homogeneity assessment

Based on the available data, ORF91a has been successfully expressed in E. coli with an N-terminal His-tag and can be purified to >90% homogeneity . The protein is typically obtained as a lyophilized powder and can be reconstituted in appropriate buffers for downstream applications.

How can the purity and activity of recombinant ORF91a be verified?

Verifying purity and activity of recombinant ORF91a requires multiple complementary approaches:

• Purity assessment methods:

  • SDS-PAGE with Coomassie staining (>90% purity standard)

  • Silver staining for detecting low-level contaminants

  • Western blotting with anti-His antibodies

  • Analytical size exclusion chromatography

  • Mass spectrometry to confirm protein identity and integrity

• Structural integrity verification:

  • Circular dichroism spectroscopy to assess secondary structure

  • Fluorescence spectroscopy to evaluate tertiary structure

  • Limited proteolysis to confirm proper folding

  • Thermal shift assays to determine stability

  • Native PAGE to assess oligomeric state

• Functional activity assays:

  • Liposome binding or integration assays

  • Membrane perturbation assays

  • Protein-protein interaction studies with candidate partners

  • Host cell binding studies if receptor interaction is suspected

  • Viral assembly assays if structural role is hypothesized

• Thermostability testing:

  • Heat treatment at various temperatures (70-90°C)

  • Activity retention after heating

  • Differential scanning calorimetry

  • Circular dichroism melting curves

  • Aggregation monitoring at elevated temperatures

• Storage stability assessment:

  • Testing various buffer conditions

  • Evaluating freeze-thaw stability

  • Monitoring activity over time at 4°C and -20°C

  • Lyophilization and reconstitution efficiency

  • Long-term stability in 50% glycerol at -80°C

According to available information, recombinant ORF91a can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and addition of 5-50% glycerol is recommended for long-term storage at -20°C/-80°C . Repeated freeze-thaw cycles should be avoided to maintain protein integrity.

What are the considerations for designing mutations in ORF91a for functional studies?

Designing mutations in ORF91a requires thoughtful strategies based on structural and functional hypotheses:

• Rational mutation design approaches:

  • Alanine scanning of predicted functional regions

  • Conservation-based mutation targets (focus on highly conserved residues)

  • Charge reversal mutations to disrupt electrostatic interactions

  • Cysteine introduction for disulfide crosslinking or labeling studies

  • Proline substitutions to disrupt helical structures

• Transmembrane domain targeting:

  • Identify key residues in predicted transmembrane regions

  • Consider helix-disrupting mutations (P, G insertions)

  • Target residues at lipid-water interfaces

  • Evaluate conserved motifs within transmembrane regions

  • Design mutations affecting predicted helix-helix interactions

• Terminal domain mutations:

  • N-terminal and C-terminal modifications based on predicted topology

  • Truncation series to identify minimal functional domains

  • Tag insertion at various positions to probe topology

  • Chimeric constructs with related viral proteins

• Mutation analysis approaches:

  • Predictive computational assessment before experimental testing

  • Stability prediction using tools like FoldX

  • Molecular dynamics simulations to assess structural impacts

  • Use homology models to guide mutation design

• Control mutations design:

  • Include conservative mutations as controls

  • Design structurally neutral mutations

  • Create mutations in non-conserved regions as negative controls

  • Include known functional mutants from related proteins if available

When designing mutations, it's important to consider the extremophilic nature of ORF91a. Mutations that might be destabilizing in mesophilic proteins may have even greater impacts in proteins adapted to extreme conditions. Similar approaches have been successfully used in other archaeal viral proteins, such as the E86A mutation in AFV1-157 that demonstrated the importance of this residue for nuclease activity .

How can the transmembrane domains of ORF91a be experimentally validated?

Experimental validation of ORF91a transmembrane domains requires multiple complementary approaches:

• Biochemical validation methods:

  • Protease protection assays using reconstituted proteoliposomes

  • Chemical labeling with membrane-impermeable reagents

  • Glycosylation mapping using engineered sites

  • FRET-based distance measurements between domains

  • Deuterium exchange mass spectrometry to identify protected regions

• Biophysical characterization approaches:

  • Circular dichroism spectroscopy in membrane-mimetic environments

  • Attenuated total reflection FTIR spectroscopy

  • Oriented circular dichroism to determine helix tilt angles

  • Solid-state NMR with isotopically labeled protein

  • EPR spectroscopy with site-directed spin labeling

• Membrane insertion assays:

  • In vitro translation in the presence of microsomes

  • Alkaline extraction to distinguish peripheral vs. integral association

  • Detergent partitioning assays

  • Fluorescence quenching experiments

  • Liposome flotation assays

• Computational validation:

  • Compare experimental results with multiple prediction algorithms

  • Molecular dynamics simulations in explicit lipid bilayers

  • Energy minimization of alternative topological models

  • Hydrophobic moment and helical wheel analysis

  • Compare with structural data from homologous proteins

• In vivo approaches:

  • Reporter fusion constructs expressed in archaeal hosts

  • Subcellular fractionation followed by immunoblotting

  • Cross-linking studies in native membranes

  • Fluorescence microscopy of tagged variants

  • Growth complementation assays with mutant variants