Recombinant His1 virus Putative transmembrane protein ORF21 (ORF21)

What is the His1 virus ORF21 protein and what is its basic structure?

His1 virus ORF21 is a putative transmembrane protein (73 amino acids) encoded by open reading frame 21 in the His1 virus genome. It is characterized by:

• Amino acid sequence: MVEIDDGVEMAVGIFVVIILAANLLPTAFDQIFNASTSSWNSDVTTLWELLPLLSVVGLLLMFVYWARKAGKM

• Predicted transmembrane domains within its structure

• Function as a major structural component in the virus capsid

• Derived from His1 virus (isolate Australia/Victoria), also known as Haloarcula hispanica virus 1

The protein contains hydrophobic domains characteristic of membrane-spanning regions, allowing it to integrate into lipid bilayers. Unlike fuselloviruses which encode two paralogous major capsid proteins (MCPs), His1 virus utilizes a single MCP which is the product of this ORF21 gene .

How does His1 virus ORF21 differ from other viral transmembrane proteins?

His1 virus ORF21 possesses several distinctive features compared to other viral transmembrane proteins:

• Evolutionary uniqueness: Unlike the bicaudaviruses which display helix bundle topology in their major capsid proteins, His1 ORF21 is characterized by two hydrophobic domains

• Structural simplicity: While fuselloviruses require two paralogous MCPs (VP1 and VP3), His1 virus functions with a single MCP (ORF21)

• Archaeal host specificity: Adapted for function in extreme halophilic environments where its host Haloarcula hispanica thrives

• Comparative analysis: His1 ORF21 shows evolutionary relationships to structural proteins in other spindle-shaped archaeal viruses, despite infecting hosts from different archaeal phyla—Crenarchaeota and Euryarchaeota

For experimental work, these differences are significant when designing comparative studies or when selecting viral proteins for structure-function analyses across different viral systems.

What are the recommended protocols for reconstitution and storage of recombinant His1 virus ORF21 protein?

For optimal handling of recombinant His1 virus ORF21 protein:

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended 50%) for long-term storage

• Aliquot to minimize freeze-thaw cycles

Storage Conditions:

• Store at -20°C/-80°C upon receipt

• For long-term storage, maintain at -20°C/-80°C in glycerol-containing buffer

• Working aliquots can be stored at 4°C for up to one week

• Storage buffer typically contains Tris/PBS-based buffer with 6% Trehalose, pH 8.0

Critical Notes:

• Repeated freezing and thawing is strongly discouraged as it compromises protein integrity

• The protein has >90% purity as determined by SDS-PAGE, which is suitable for most research applications

• If precipitation occurs after thawing, gentle mixing or brief sonication may help resolubilize the protein

How can researchers verify the functional activity of recombinant His1 virus ORF21 protein in experimental systems?

Verification of recombinant His1 virus ORF21 functionality requires multiple complementary approaches:

Structural Integrity Assessment:

• Circular dichroism (CD) spectroscopy: Monitor secondary structure elements to confirm proper folding

• Size exclusion chromatography: Verify monomeric state or oligomerization status

• Thermal shift assays: Assess protein stability under various buffer conditions

Functional Characterization:

• Lipid bilayer integration assays: Since ORF21 is a putative transmembrane protein, verify membrane insertion using liposome flotation assays or fluorescence-based membrane integration approaches

• Virus-like particle (VLP) formation: Test incorporation into artificial lipid membranes or virus-like particles

• Protein-protein interaction studies: Investigate interactions with other viral components using pull-down assays or surface plasmon resonance

Comparison to Native Context:

• Electron microscopy: Compare structures of assembled particles with recombinant ORF21 to wild-type viral structures

• Immunolocalization: Use antibodies against ORF21 to verify localization in reconstituted systems matches native viral distribution

When assessing activity, researchers should consider that as a structural protein, "function" may be defined by correct assembly and interaction properties rather than enzymatic activity.

What experimental approaches can be used to study the membrane topology of His1 virus ORF21 protein?

Several complementary approaches can determine the membrane topology of ORF21:

Computational Prediction Methods:

• Hydropathy analysis: Using algorithms like TMHMM or Kyte-Doolittle plots to predict transmembrane regions

• Topology prediction software: Tools like TOPCONS or MEMSAT to predict orientation of transmembrane segments

Biochemical Approaches:

• Protease protection assays: Limited proteolysis of ORF21 in reconstituted liposomes to identify exposed regions

• Chemical modification: Using membrane-impermeable reagents to modify accessible amino acid residues

• Glycosylation mapping: Introduction of artificial glycosylation sites to determine lumenal vs. cytoplasmic orientation

Biophysical Methods:

• Fluorescence spectroscopy: Site-specific labeling of ORF21 with fluorophores to monitor membrane insertion

• FRET analysis: Measuring distances between donor-acceptor pairs placed at strategic positions

• Cryo-electron microscopy: Visualizing the protein in reconstituted membranes or virus-like particles

Expression Systems:

• In vitro translation: Cell-free systems with microsomes to analyze membrane insertion

• Reporter fusion proteins: Creating ORF21 fusions with reporters like GFP or PhoA to determine orientation

Based on current data, ORF21 likely contains multiple transmembrane regions, making it important to use complementary techniques to build a complete topological model .

How does His1 virus ORF21 relate to structural proteins in other archaeal viruses?

His1 virus ORF21 shows important evolutionary relationships with other archaeal viral structural proteins:

Fuselloviruses Comparison:

Evolutionary Positioning:

• His1 virus ORF21 represents a case of evolutionary conservation of capsid protein structure across archaeal phyla divisions

• In the taxonomic context, His1 virus belongs to the group of tailless spindle-shaped viruses related to the Fuselloviridae family

• Metagenomic studies suggest ORF21-like proteins may be widespread in extreme environments

Structural Role:

• His1 ORF21 functions as a major capsid protein, similar to VP21 in related viruses

• The single MCP strategy of His1 contrasts with the dual MCP strategy (VP1 and VP3) employed by fuselloviruses, representing different evolutionary solutions to capsid assembly

This comparative context is valuable for researchers studying viral evolution and archaeal virus structural biology.

What insights can be gained from studying His1 virus ORF21 in relation to PH1 and SH1 viruses?

Comparative analysis of His1 virus ORF21 with similar proteins in PH1 and SH1 viruses provides several valuable insights:

Structural Conservation:

• Based on mass spectrometry analysis of PH1, the corresponding structural protein VP4 is encoded by ORF21, suggesting functional equivalence across these related viruses

• The similarity between these proteins helps establish evolutionary relationships among haloarchaeal viruses

Genomic Context:

• PH1 and SH1 show close similarity (74% nucleotide identity), allowing detailed analysis of divergent regions that may affect protein function

• Comparative genomics reveals extensive recombination between known haloviruses, which may influence ORF21 evolution

Functional Implications:

• Studies of SH1-like and pleolipoviruses in relation to previously described genomic loci of virus and plasmid-related elements (ViPREs) of haloarchaea revealed extensive recombination events

• This suggests ORF21 exists within a dynamic genomic context that may influence its evolutionary trajectory

Research Applications:

• Experimental approaches that successfully characterized PH1 virion proteins (including separation by gel electrophoresis, digestion with trypsin, and analysis by MALDI-TOF MS) can be applied to study His1 ORF21

• The His1-PH1-SH1 relationship offers a model system for studying viral protein evolution in extreme environments

These comparisons help place His1 virus ORF21 within the broader context of archaeal virus evolution and structural biology.

How can structural studies of His1 virus ORF21 contribute to understanding archaeal virus assembly?

Structural investigations of His1 virus ORF21 provide critical insights into archaeal virus assembly mechanisms:

Cryo-EM and X-ray Crystallography Applications:

• High-resolution structural determination of ORF21 can reveal:

  • Domain organization and secondary structure elements

  • Potential oligomerization interfaces

  • Membrane interaction surfaces

  • Conformational changes during assembly

• Integration with techniques such as 3D correlative structured illumination microscopy can visualize assembly intermediates

Structure-Function Relationships:

• Mapping functional domains through targeted mutagenesis and correlating with structural features

• Identifying conserved structural motifs that might be present in other capsid proteins despite sequence divergence

• Understanding how a single MCP (ORF21) performs functions that require two MCPs in related viruses

Virus Assembly Models:

• ORF21 structure can inform models of spindle-shaped virus assembly

• Understanding how archaeal lipids interact with the transmembrane domains of ORF21

• Insights into adaptations for extreme halophilic environments

Methodological Considerations:

• Recombinant ORF21 can be used to reconstitute virus-like particles under controlled conditions

• Comparing assembly kinetics and efficiency between His1 ORF21 and other archaeal viral capsid proteins

• Using high-salt conditions that mimic the native environment of halophilic archaea

These structural studies are fundamental to understanding the unique morphology of spindle-shaped archaeal viruses and their assembly pathways.

What analytical techniques are most effective for studying interactions between His1 virus ORF21 and viral or host membranes?

Several specialized techniques can effectively investigate ORF21-membrane interactions:

Biophysical Approaches:

• Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR): Measures secondary structure changes upon membrane binding

• Surface Plasmon Resonance (SPR): Quantifies binding kinetics to immobilized lipid layers

• Quartz Crystal Microbalance with Dissipation monitoring (QCM-D): Measures mass and viscoelastic properties during membrane insertion

• Atomic Force Microscopy (AFM): Visualizes topographical changes in membranes upon ORF21 integration

Fluorescence-Based Methods:

• Fluorescence Correlation Spectroscopy (FCS): Measures diffusion coefficients of labeled ORF21 in membranes

• Förster Resonance Energy Transfer (FRET): Detects proximity between labeled ORF21 and membrane components

• Fluorescence Recovery After Photobleaching (FRAP): Quantifies lateral mobility of ORF21 in membranes

Model Membrane Systems:

• Liposomes with archaeal lipid composition: Mimics the native viral environment

• Giant Unilamellar Vesicles (GUVs): Allows visualization of protein-induced membrane deformations

• Supported lipid bilayers: Enables surface-sensitive techniques like Total Internal Reflection Fluorescence (TIRF)

Molecular Dynamic Simulations:

• Computational modeling of ORF21 insertion into archaeal lipid bilayers

• Prediction of protein-lipid interactions and conformational changes

When designing these experiments, researchers should consider the unique lipid composition of archaeal membranes and the high salt conditions required for proper folding of halophilic proteins.

How might researchers apply knowledge from herpesvirus ORF21 studies to investigate His1 virus ORF21 functions?

While His1 virus ORF21 and herpesvirus ORF21 are not homologous proteins, research approaches can be translated between these systems:

Translatable Methodological Approaches:

• Genetic manipulation strategies: The generation of ORF21-mutated KSHV BAC clones (kinase-deficient and deletion mutants) demonstrates an approach that could be adapted to archaeal systems

• Comparative functional analysis: Testing multiple functions (similar to how KSHV ORF21 was analyzed for both enzymatic and non-enzymatic roles)

• Temporal expression analysis: Determining when during viral replication ORF21 is expressed, as done with KSHV ORF21 (noted to be expressed 36h after lytic induction)

Functional Investigation Frameworks:

• Protein-protein interaction networks: Identifying interaction partners of His1 ORF21 using approaches like co-immunoprecipitation and mass spectrometry

• Subcellular localization studies: Determining localization patterns during viral replication

• Host-factor interactions: Investigating whether His1 ORF21 manipulates any host cell functions

Signal Pathway Analysis:

• While KSHV ORF21 affects MEK phosphorylation , His1 ORF21 may interact with archaeal signaling systems

• Exploring whether His1 ORF21 modulates host cell processes beyond structural roles

Transcriptional Regulation:

• The KSHV ORF21 promoter contains specific response elements (XREs) ; investigating whether His1 ORF21 expression is regulated by specific archaeal transcription factors

These translatable approaches can help expand our understanding of His1 virus ORF21 functions, potentially revealing roles beyond its established structural function.

What are the major challenges in expressing and purifying functional His1 virus ORF21 protein, and how can they be addressed?

Researchers face several challenges when working with His1 virus ORF21 protein:

Expression Challenges and Solutions:

Purification Strategies:

• Two-step purification protocol: IMAC (using His-tag) followed by size exclusion chromatography

• On-column refolding: Gradual removal of denaturant during affinity purification

• Validation of oligomeric state: Use analytical ultracentrifugation to confirm proper assembly

Quality Control Measures:

• Circular dichroism: Verify secondary structure formation

• Dynamic light scattering: Assess homogeneity and aggregation state

• Thermal shift assays: Identify optimal buffer conditions

Reconstitution Approaches:

• Stepwise dialysis: Gradual removal of detergent in presence of lipids

• Direct incorporation: Addition of protein during liposome formation

• High-salt conditions: Maintain halophilic conditions (2-4M salt) throughout purification

These strategies can significantly improve the yield and quality of recombinant His1 virus ORF21 protein for functional and structural studies.

How can researchers design experiments to investigate the role of His1 virus ORF21 in viral assembly and infection?

A comprehensive experimental approach to investigate His1 virus ORF21 function includes:

Genetic Approaches:

• CRISPR-Cas9 editing of viral genome: Generate ORF21 deletions or point mutations

• Complementation assays: Express wild-type ORF21 in trans to rescue mutant phenotypes

• Domain swapping: Replace regions of ORF21 with corresponding regions from related viruses

Structural Assembly Studies:

• Electron microscopy: Compare morphology of wild-type and ORF21-mutant virions

• In vitro assembly assays: Reconstitute capsid formation using purified components

• Pulse-chase experiments: Track the incorporation of ORF21 into assembling virions

Infection Models:

• Single-step growth curves: Compare replication kinetics between wild-type and mutant viruses

• Attachment and entry assays: Determine if ORF21 affects early infection events

• Fluorescence microscopy: Visualize infection progression using fluorescently labeled virions

Protein-Protein Interaction Studies:

• Proximity labeling: Identify proteins that interact with ORF21 during infection

• Co-immunoprecipitation: Verify specific interactions with other viral proteins

• Yeast two-hybrid screening: Identify potential host interaction partners

Experimental Controls:

• Parallel studies with related viruses: Compare with PH1 and SH1 viral systems

• Host range analysis: Test infection of different Haloarcula species

• Complementation with heterologous capsid proteins: Test functional conservation

This multifaceted approach can provide comprehensive insights into the role of ORF21 in the viral life cycle while addressing the technical challenges of working with archaeal systems.

What emerging technologies might advance our understanding of His1 virus ORF21 structure and function?

Several cutting-edge technologies show promise for elucidating ORF21 biology:

Advanced Structural Biology Approaches:

• Cryo-electron tomography: Visualize ORF21 organization within intact virions at molecular resolution

• Microcrystal electron diffraction (MicroED): Determine structures from nano-sized crystals of membrane proteins

• Correlative light and electron microscopy (CLEM): Integrate functional and structural imaging

• Single-particle cryo-EM with membrane proteins: Resolve ORF21 structure in native-like lipid environments

Functional Genomics Technologies:

• CRISPR interference in archaeal systems: Precise modulation of ORF21 expression

• High-throughput mutagenesis with deep mutational scanning: Comprehensive functional mapping

• Synthetic biology approaches: Bottom-up reconstruction of minimal viral systems

Mass Spectrometry Innovations:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Probe structural dynamics and conformational changes

• Crosslinking mass spectrometry (XL-MS): Map interaction interfaces within viral particles

• Native mass spectrometry: Analyze intact complexes containing ORF21

Advanced Imaging:

• Super-resolution microscopy: Track single ORF21 molecules during virion assembly

• Lattice light-sheet microscopy: Image virus assembly dynamics in live archaeal cells

• 4D electron microscopy: Capture structural transitions during assembly processes

Computational Methods:

• AlphaFold2 and RoseTTAFold: Predict ORF21 structure with improved accuracy

• Molecular dynamics simulations: Model membrane interactions in archaeal lipid environments

• Machine learning classification: Identify subtle patterns in ORF21 sequence-structure relationships

These technologies can collectively address the current limitations in studying the structure-function relationships of archaeal viral proteins in their native contexts.

How might research on His1 virus ORF21 contribute to broader understanding of viral evolution across extreme environments?

Research on His1 virus ORF21 offers unique perspectives on viral adaptation and evolution:

Evolutionary Insights:

• Archaeal virus phylogeny: His1 ORF21 provides evidence for evolutionary relationships between viruses infecting different archaeal phyla

• Convergent evolution: Comparison with other viral transmembrane capsid proteins can reveal independent solutions to similar structural challenges

• Host-virus co-evolution: Understanding how ORF21 adapted to function in extreme halophilic environments

Extremophile Virology:

• Adaptation mechanisms: Identifying molecular features that allow ORF21 to function in high-salt environments

• Structural stability: Understanding how capsid proteins maintain integrity under extreme conditions

• Comparative analysis: Contrasting His1 ORF21 with viral proteins from thermophilic, acidophilic, and psychrophilic environments

Broader Implications:

• Ancient viral lineages: His1 virus may represent ancestral viral forms that predate the divergence of archaeal phyla

• Archaeal virus classification: Contributes to developing taxonomy based on protein structural relationships rather than sequence similarity alone

• Horizontal gene transfer: Investigating the role of recombination in shaping His1 ORF21 evolution, similar to patterns observed in related viruses

Applications:

• Bionanotechnology: Engineering stable protein assemblies based on extremophile viral proteins

• Astrobiology: Understanding viral adaptation to extreme conditions relevant to potential extraterrestrial environments

• Synthetic biology: Designing proteins that function under non-standard conditions

This research contributes to fundamental questions about viral origin, diversity, and adaptation while providing practical insights for biotechnology applications.

What are the recommended approaches for studying the biophysical properties of His1 virus ORF21 in high-salt environments?

Studying ORF21 in high-salt conditions requires specialized approaches:

Buffer Considerations:

• Salt composition: Use buffers containing 2-4M NaCl or mixed salts (NaCl, KCl, MgCl₂) mimicking the Dead Sea or salterns

• pH stability: Select buffers with minimal pH variation in high-salt conditions (PIPES, HEPES)

• Protein stabilizers: Include osmolytes like glycerol (5-10%) and trehalose (100-200 mM)

Membrane Mimetic Systems:

• Archaeal lipid extracts: Use lipids from Haloarcula or synthetic archaeal-like lipids

• Nanodiscs with salt-stable membrane scaffold proteins: For single-particle studies

• Amphipol A8-35: Stabilizes membrane proteins in high salt without detergents

Biophysical Techniques Modified for High Salt:

• Differential scanning calorimetry (DSC): Measure thermal stability in high-salt conditions

• Circular dichroism with short path-length cells: Reduce signal-to-noise issues from high salt

• Size exclusion chromatography with salt-stable columns: Monitor oligomeric state

Specialized Analytical Methods:

• Small-angle neutron scattering (SANS): Especially powerful for high-salt solutions

• Isothermal titration calorimetry (ITC): Measure binding interactions in native-like conditions

• Halophile-adapted fluorescence approaches: Account for salt effects on fluorophores

Experimental Controls:

• Salt-dependent measurements: Perform experiments across salt gradients (1-4M)

• Comparison with mesophilic proteins: Include non-halophilic controls to benchmark salt effects

• Multiple salt types: Test effects of different ionic compositions (Na⁺, K⁺, Mg²⁺)

These specialized approaches allow researchers to study ORF21 under conditions that maintain its native structure and function.

What analytical methods can determine whether recombinant His1 virus ORF21 correctly folds and assembles compared to the native viral protein?

Multiple complementary analytical approaches can verify proper folding and assembly:

Structural Comparison Methods:

• Circular dichroism spectroscopy: Compare secondary structure profiles between recombinant and native proteins

• FTIR spectroscopy: Assess secondary structure elements with high sensitivity to β-sheet content

• Limited proteolysis: Compare digestion patterns as indicators of similar folding

• Intrinsic fluorescence spectroscopy: Measure exposure of tryptophan residues

Functional Comparison Approaches:

• Membrane integration assays: Verify similar patterns of membrane association

• Assembly competence: Test ability to form virus-like particles with other viral components

• Antibody recognition: Use conformation-specific antibodies to verify structural epitopes

Advanced Structural Analysis:

• Negative-stain electron microscopy: Compare morphology of assemblies

• Hydrogen-deuterium exchange mass spectrometry: Map solvent accessibility profiles

• Small-angle X-ray scattering (SAXS): Compare solution structures and conformational ensembles

Quality Assessment Metrics: