Recombinant Acidianus filamentous virus 1 Uncharacterized protein ORF55 (ORF55)

What is the genomic context of ORF55 within the AFV1 genome, and how does this inform potential functional predictions?

The ORF55 gene is one of 40 open reading frames identified in the 20,869 bp genome of AFV1 . While specific information about ORF55's genomic context is limited in current research, viral gene organization often provides clues to function. In related archaeal viruses, genes encoding proteins with similar functions are frequently clustered together.

Methodological approach: To analyze ORF55's genomic context:

• Perform comparative genomic analysis using related Lipothrixviridae genomes

• Identify syntenic regions where gene order is conserved

• Analyze the functions of neighboring genes, as they may participate in similar processes

• Examine upstream regulatory sequences for potential co-expression patterns with functionally characterized genes

How does the extreme environment of the host Acidianus hospitalis influence approaches to studying AFV1 proteins like ORF55?

Acidianus hospitalis thrives in acidic hot springs at temperatures above 85°C and pH ≈1.5 . These extreme conditions have significant implications for studying AFV1 proteins:

Methodological considerations:

• Proteins must be expressed in systems capable of producing thermostable proteins with correct folding

• Standard biochemical assays may require modification to accommodate extreme pH and temperature conditions

• Structural studies must account for the protein's native thermostable configuration

• Functional assays should ideally mimic the extreme conditions of the natural host environment

When designing experiments to study ORF55, researchers should incorporate buffers and conditions that reflect the acidophilic, hyperthermophilic native environment to ensure relevant results .

What can sequence analysis reveal about the potential structure and function of ORF55?

While the search results don't provide specific information about ORF55's sequence, methodological approaches for sequence analysis of uncharacterized archaeal viral proteins typically include:

• Homology detection using sensitive methods like PSI-BLAST, HHpred, or HMMER to identify distant relationships

• Secondary structure prediction using tools like PSIPRED or JPred

• Identification of conserved domains using InterPro, Pfam, or CDD

• Analysis for structural motifs common in DNA/RNA binding proteins (if relevant)

• Prediction of intrinsically disordered regions which may suggest flexible binding interfaces

For thermostable proteins like those in AFV1, additional analysis for features associated with thermostability (increased ionic interactions, hydrophobic cores, reduced loop regions) can provide insight into structure .

What expression systems are most appropriate for recombinant production of hyperthermophilic viral proteins like ORF55?

Based on successful expression of other AFV1 proteins, the following methodological approaches are recommended:

Expression system considerations:

• E. coli-based expression: As demonstrated with AFV1-132 and AFV1-140, E. coli can be used for expression of archaeal viral proteins . Consider codon optimization and use of specialized strains designed for challenging proteins.

• Archaeal expression systems: For proteins that fail to express functionally in bacterial systems, consider homologous or heterologous archaeal expression systems based on Sulfolobus or related species.

• Fusion tags selection: For hyperthermophilic proteins:

  • Thermostable tags like thermostable GFP variants

  • Maltose-binding protein (MBP) for improved solubility

  • His-tags placed at C-terminus rather than N-terminus if N-terminal structure is critical

• Temperature considerations: Expression at elevated temperatures (30-37°C) may improve folding of thermophilic proteins in mesophilic hosts .

What purification challenges are specific to recombinant archaeal virus proteins, and how can they be addressed?

Purification of archaeal viral proteins presents unique challenges:

Methodological solutions:

• Heat treatment: Exploit the thermostability of the target protein by heating cell lysates (60-80°C) to precipitate less stable host proteins, as likely performed for AFV1 proteins .

• pH considerations: Incorporate pH adjustments that reflect the acidophilic nature of the native environment (pH 3-5).

• Buffer optimization:

  • Include stabilizing agents like glycerol or specific ions (Mg²⁺, Ca²⁺) as used in AFV1-140 DNA binding studies

  • Test buffers containing 12.8 mM CaCl₂, 12.8 mM MgCl₂, and 1.28 mM NaN₃ as used successfully for AFV1 proteins

• Specialized chromatography: Hydrophobic interaction chromatography may be particularly effective for proteins with amphiphilic properties similar to AFV1-140 .

What structural biology approaches are most informative for characterizing ORF55?

Based on successful structural studies of other AFV1 proteins, consider these methodological approaches:

• X-ray crystallography: Successful for AFV1-132 and AFV1-140, with crystals grown at room temperature using the hanging-drop vapor-diffusion method . Apply similar conditions using:

  • 0.1 M Tris–HCl pH 8.5

  • 0.2 M MgCl₂

  • 30% PEG4000

  • Microseeding techniques to improve crystal quality

• Cryo-electron microscopy: Particularly valuable if ORF55 forms filamentous structures like the major coat proteins or participates in larger complexes .

• NMR spectroscopy: For smaller domains or if crystallization proves challenging.

• Small-angle X-ray scattering (SAXS): To obtain low-resolution structural information in solution.

• Circular dichroism (CD): To rapidly assess secondary structure content and thermal stability .

How might ORF55 interact with AFV1's genomic DNA or other viral proteins?

The major coat proteins of AFV1 (AFV1-132 and AFV1-140) bind DNA and form filaments with linear dsDNA . If ORF55 has similar properties, consider these methodological approaches:

• DNA binding assays:

  • Electrophoretic mobility shift assays (EMSA) as performed with AFV1-132 and AFV1-140

  • Fluorescence anisotropy with labeled DNA fragments

  • Surface plasmon resonance (SPR) to quantify binding kinetics

• Protein-protein interaction studies:

  • Co-immunoprecipitation with other AFV1 proteins

  • Yeast two-hybrid adapted for thermophilic proteins

  • Cross-linking mass spectrometry to capture transient interactions

  • Proximity labeling in reconstituted systems

• Filament formation assessment:

  • Negative-stain electron microscopy using conditions similar to those for AFV1 MCPs (incubation with linear dsDNA at 60-78°C)

  • Test various buffer conditions, including those with divalent cations (Ca²⁺, Mg²⁺)

• In silico docking: Computational prediction of DNA interaction sites based on electrostatic surface potential .

What approaches can determine if ORF55 contributes to the unique lipid-containing envelope of AFV1?

The AFV1 virion features a lipid-containing outer shell, with AFV1-140 potentially forming part of this envelope through its amphiphilic C-terminal helix . To investigate if ORF55 plays a similar role:

Methodological approaches:

• Sequence analysis: Search for amphiphilic helices or hydrophobic regions that might interact with lipids.

• Lipid binding assays:

  • Liposome flotation assays

  • Monolayer penetration experiments

  • Lipid overlay assays (PIP strips)

• Structural characterization in membrane-mimetic environments:

  • Protein crystallization in the presence of detergents

  • NMR with nanodiscs or bicelles

  • Cryo-EM of reconstituted protein-lipid complexes

• Biophysical characterization:

  • Differential scanning calorimetry to detect lipid interactions

  • Circular dichroism to assess structural changes upon lipid binding

What bioinformatic approaches can predict function when sequence homology is limited?

For uncharacterized proteins like ORF55 with limited sequence homology, advanced computational methods are essential:

Methodological framework:

• Structure prediction using deep learning:

  • AlphaFold2 or RoseTTAFold for ab initio structural models

  • Comparison of predicted structures to known folds in structural databases

• Threading approaches:

  • I-TASSER, PHYRE2, or SWISS-MODEL to detect structural similarities

  • Structural classification to identify potential functions

• Genomic context analysis:

  • Gene neighborhood conservation across related viruses

  • Co-evolution analysis to identify potential interaction partners

• Machine learning integration:

  • Feature extraction combining sequence, predicted structure, and genomic context

  • Supervised machine learning using known archaeal viral proteins as training data

How should researchers analyze and interpret structural data for ORF55 in the context of related archaeal viral proteins?

The structural similarity between AFV1 coat proteins and those of SIRV (Sulfolobus islandicus rod-shaped virus) despite low sequence identity (13%) demonstrates the importance of structural comparison . For ORF55:

Methodological framework:

• Structural comparison pipeline:

  • DALI or FATCAT for detecting structural similarities

  • TM-align for alignment of predicted or experimental structures

  • Analysis of conserved structural motifs rather than sequence

• Evolutionary interpretation:

  • Place findings in the context of the proposed Ligamenvirales order that groups Lipothrixviridae and Rudiviridae

  • Consider if structural similarities support vertical inheritance from a common viral ancestor

• Structure-function correlation:

  • Map conserved residues onto structural models

  • Identify potential functional sites through electrostatic analysis

  • Compare with the four-helix bundle topology found in AFV1 MCPs and SIRV coat protein

What considerations are important when interpreting protein-protein or protein-DNA interaction data for ORF55?

When studying interactions involving uncharacterized proteins like ORF55, careful data interpretation is critical:

Methodological considerations:

• False positive filtration:

  • Implement stringent controls for non-specific binding

  • Validate interactions using multiple orthogonal methods

  • Consider the extreme conditions of the native environment

• Biological relevance assessment:

  • Test interactions under conditions mimicking the viral environment (high temperature, low pH)

  • Compare binding affinities with those of known functional interactions

  • Assess conservation of interaction interfaces across related viruses

• Integration with virion structural models:

  • Evaluate how identified interactions might contribute to the AFV1 filamentous structure

  • Compare with the proposed model where DNA wraps around basic proteins while amphiphilic domains interact with the lipid envelope

How might research on ORF55 contribute to understanding archaeal virus evolution?

The structural similarity between proteins from Lipothrixviridae (AFV1) and Rudiviridae (SIRV) supports their classification in the Ligamenvirales order . Studies of ORF55 could further inform viral evolution:

Methodological approaches:

• Comparative structural genomics:

  • Systematically characterize structures of uncharacterized proteins across archaeal viruses

  • Build structural phylogenies to complement sequence-based approaches

  • Identify conserved structural features that may represent ancient viral hallmark proteins

• Ancestral sequence reconstruction:

  • Infer ancestral sequences of ORF55-like proteins

  • Express and characterize these reconstructed proteins

  • Map the evolutionary trajectory of structural and functional changes

• Host-virus co-evolution studies:

  • Investigate adaptation signatures in viral proteins to different archaeal hosts

  • Examine how extreme environments shape viral protein evolution

What emerging technologies might advance the study of uncharacterized archaeal virus proteins like ORF55?

Novel methodological approaches for studying challenging proteins include:

• Nanopore sensing:

  • Direct detection of protein-DNA interactions at the single-molecule level

  • Real-time monitoring of binding events under extreme conditions

• Cryo-electron tomography:

  • Visualize ORF55 in the context of the intact virion

  • Generate 3D reconstructions of the viral architecture

• In situ structural biology:

  • Time-resolved studies using X-ray free-electron lasers (XFELs)

  • Visualize conformational changes during viral assembly

• Synthetic biology approaches:

  • Minimal reconstitution of viral assembly systems

  • Design of artificial proteins based on archaeal virus structural motifs

  • Engineering of thermostable proteins for biotechnological applications