Recombinant His1 virus Putative transmembrane protein ORF25 (ORF25)

How is recombinant His1 virus ORF25 protein typically produced for research purposes?

Recombinant His1 virus ORF25 protein is typically produced using bacterial expression systems, primarily E. coli, with an N-terminal His tag to facilitate purification . The methodological approach involves:

• Gene synthesis or cloning of the ORF25 gene into an appropriate expression vector

• Transformation of the vector into a suitable E. coli strain (commonly BL21(DE3) or derivatives)

• Induction of protein expression, typically using IPTG for T7-based expression systems

• Cell lysis under conditions that maintain protein structure

• Purification using nickel affinity chromatography to capture the His-tagged protein

• Further purification steps such as size exclusion chromatography if higher purity is required

The expression of membrane proteins like ORF25 often presents challenges due to potential toxicity and inclusion body formation. Strategies to address these challenges include:

• Using lower induction temperatures (16-25°C)

• Employing specialized E. coli strains designed for membrane protein expression

• Adding solubility-enhancing fusion partners

• Optimizing expression media composition

What structural features characterize the ORF25 protein and how might they relate to function?

The ORF25 protein contains several key structural features that provide insights into its potential functions:

Secondary structure prediction suggests the protein likely adopts an alpha-helical conformation in membrane-spanning regions with possible beta-turns at interface regions. This structural arrangement is consistent with proteins that function in membrane remodeling during viral replication, similar to the role of certain proteins in other viral systems .

The amino acid composition, particularly the high content of hydrophobic residues (leucine, isoleucine, phenylalanine) supports membrane integration, while the presence of glycine and serine residues may provide the flexibility needed for membrane curvature induction .

How does ORF25 compare to transmembrane proteins from other archaeal viruses?

When comparing ORF25 to transmembrane proteins from other archaeal viruses, several patterns emerge:

• Size comparison: At 93 amino acids, ORF25 is relatively small compared to many viral transmembrane proteins, suggesting it may function as part of a larger protein complex rather than independently

• Sequence conservation: ORF25 shows limited sequence homology with other archaeal viral proteins, but structural predictions suggest functional convergence with other viral transmembrane proteins

• Evolutionary considerations: The unique characteristics of ORF25 may reflect adaptation to the extreme halophilic environment in which His1 virus replicates

• Functional parallels: Despite sequence divergence, ORF25 likely performs functions similar to membrane-associated proteins from other viruses, such as membrane remodeling for replication complex formation or virion assembly and egress

This comparative analysis highlights the evolutionary diversity of archaeal viruses while suggesting functional conservation of certain protein roles. The adaptation to extreme environments may drive unique structural solutions to common viral requirements for membrane manipulation .

What are the optimal conditions for reconstitution and storage of purified recombinant ORF25 protein?

Proper reconstitution and storage of purified recombinant ORF25 protein is critical for maintaining its structural integrity and functionality. Based on experimental data, the following methodology is recommended:

For reconstitution:

• Centrifuge the lyophilized protein vial briefly to ensure all material is at the bottom

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

• For membrane proteins like ORF25, consider adding mild detergents (0.1% DDM or 0.5% CHAPS) to maintain solubility

• Allow complete dissolution by gentle mixing rather than vortexing, which can cause protein denaturation

For storage:

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

• Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles

• Store aliquots at -20°C for short-term or -80°C for long-term storage

• For working stocks, store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

Additionally, when working with transmembrane proteins like ORF25, consider buffer optimization:

• Buffer pH between 7.5-8.0 (Tris/PBS-based buffer is commonly used)

• Addition of stabilizing agents such as trehalose (6%) can significantly improve stability

• For functional studies, consider reconstitution into liposomes or nanodiscs to maintain native membrane environment

What tagging strategies are most effective for subcellular localization studies of ORF25?

When designing experiments to study the subcellular localization of His1 virus ORF25 protein, careful consideration of tagging strategies is essential. Based on successful approaches with other viral transmembrane proteins, the following methodological framework is recommended:

• Tag selection considerations:

  • Small epitope tags (HA, FLAG, Myc) are preferable for transmembrane proteins as they minimize structural disruption

  • The N-terminus appears more tolerant to tagging based on recombinant protein expression success

  • C-terminal tagging should be approached cautiously as it may interfere with potential protein-protein interactions

• Tag insertion methodology:

  • For transmembrane proteins like ORF25, insertion sites should be carefully selected to avoid disrupting membrane-spanning domains

  • A transposon-mediated random insertion approach followed by functional selection (similar to that used for HEV ORF1 ) can identify permissive insertion sites

  • Alternatively, structure-guided rational design based on predicted loop regions can be employed

• Visualization techniques:

  • Indirect immunofluorescence using tag-specific antibodies

  • Combination with fluorescence in situ hybridization (FISH) for viral RNA can identify potential replication sites, as demonstrated with tagged HEV genomes

  • Super-resolution microscopy (STORM, PALM) for precise localization within membrane compartments

• Validation approaches:

  • Functional assays to ensure tagged protein retains activity

  • Comparison of multiple tagging positions to confirm consistent localization patterns

  • Complementary approaches such as subcellular fractionation followed by Western blotting

This systematic approach allows reliable determination of ORF25 subcellular localization while minimizing artifacts associated with protein tagging. The method has been successfully applied to other viral transmembrane proteins and can be adapted for ORF25 studies .

How can protein-protein interactions involving ORF25 be effectively studied?

Investigating protein-protein interactions involving membrane proteins like ORF25 requires specialized approaches to accommodate their hydrophobic nature. A comprehensive methodology includes:

• Co-immunoprecipitation adaptations:

  • Use of crosslinking agents (DSP, formaldehyde) to stabilize transient interactions

  • Optimization of detergent conditions to solubilize membrane complexes without disrupting interactions

  • Two-step purification using differentially tagged proteins to reduce false positives

  • Controls should include antibody-only precipitations and non-specific tag controls

• Proximity-based labeling approaches:

  • BioID or TurboID fusion constructs express ORF25 fused to a biotin ligase

  • The ligase biotinylates proteins in close proximity to ORF25 in living cells

  • Biotinylated proteins are purified using streptavidin and identified by mass spectrometry

  • This approach is particularly valuable for transmembrane proteins as it does not require solubilization of complexes

• Split-reporter complementation assays:

  • ORF25 is fused to one half of a reporter protein (luciferase, fluorescent protein)

  • Potential interaction partners are fused to the complementary half

  • Interaction brings the two halves together, restoring reporter activity

  • This approach allows visualization of interactions in living cells

• Membrane yeast two-hybrid systems:

  • Modified yeast two-hybrid specifically designed for membrane proteins

  • ORF25 is expressed as a bait fused to a transcription factor's DNA-binding domain

  • Both proteins are directed to the membrane, allowing interaction detection in a membrane environment

These methodologies have been successfully applied to study interactions of viral membrane proteins similar to ORF25, revealing their roles in replication complex formation and virus assembly. For example, similar approaches with HEV proteins identified interactions with host proteins involved in the exosomal pathway .

What are the methodological considerations for functional studies of ORF25 in viral replication?

To investigate the functional role of ORF25 in His1 virus replication, a systematic experimental approach combining genetic and biochemical methods is required:

• Genetic manipulation strategies:

  • Site-directed mutagenesis to alter specific residues based on sequence conservation

  • Deletion analysis to identify essential regions within the protein

  • Domain swapping with related viral proteins to determine region-specific functions

  • For transmembrane proteins, hydrophobic residues can be replaced with alanine to disrupt membrane interactions while maintaining protein structure

• Replication assay design:

  • Development of reporter-based replication systems (similar to HEV luciferase-based systems )

  • Quantitative PCR to measure viral RNA levels following mutation of ORF25

  • Single-cell analysis techniques to capture heterogeneity in replication efficiency

  • Time-course experiments to distinguish effects on early versus late replication events

• Membrane association analysis:

  • Subcellular fractionation to determine membrane association patterns

  • Protease protection assays to map topology of ORF25 within membranes

  • Liposome binding assays to assess direct membrane interaction capabilities

  • Electron microscopy to visualize membrane alterations induced by ORF25

• Host factor identification:

  • CRISPR screens to identify host factors required for ORF25 function

  • Proximity labeling combined with mass spectrometry to identify proteins in the vicinity of ORF25 during infection

  • RNA-protein crosslinking assays to test potential interactions with viral RNA

These methodological approaches, when combined, can provide comprehensive insights into the functional role of ORF25 in viral replication. Similar strategies applied to other viral transmembrane proteins have successfully identified roles in replication complex formation, membrane remodeling, and viral assembly .

How can structural studies of ORF25 be approached given the challenges of membrane protein crystallography?

Structural characterization of membrane proteins like ORF25 presents significant challenges that require specialized methodological approaches:

• Sample preparation optimization:

  • Detergent screening is critical - start with a panel including DDM, LMNG, CHAPS, and GDN

  • Lipid cubic phase crystallization often yields better results for small membrane proteins

  • Addition of lipids during purification can stabilize native conformations

  • Nanodiscs or amphipols can maintain native-like membrane environments

• Cryo-electron microscopy approaches:

  • Single-particle cryo-EM is increasingly successful for smaller membrane proteins

  • Use of Fab fragments as fiducial markers can facilitate particle alignment

  • Sample vitrification conditions must be optimized for detergent-containing samples

  • Data processing should account for preferred orientations common with membrane proteins

• NMR-based strategies:

  • Solution NMR using detergent micelles works well for smaller membrane proteins like ORF25

  • Selective isotope labeling (15N, 13C) enhances spectral resolution

  • Solid-state NMR provides an alternative when solution NMR is challenging

  • Magic angle spinning techniques can provide atomic resolution in membrane environments

• Computational structure prediction:

  • Recent advances in AI-based structure prediction (AlphaFold, RoseTTAFold) show promise

  • Homology modeling based on structurally characterized viral membrane proteins

  • Molecular dynamics simulations to refine models and study dynamics in membrane environments

  • Validation using limited experimental constraints from crosslinking or spectroscopic data

These approaches have been successfully applied to other viral membrane proteins of similar size to ORF25. For example, structural studies of viral membrane proteins have revealed how they form pores, induce membrane curvature, or interact with host proteins - all potential functions of ORF25 in the His1 virus life cycle .

What approaches can be used to investigate the potential role of ORF25 in viral entry or assembly?

Investigating the role of ORF25 in viral entry or assembly requires a multifaceted experimental approach:

• Virus-like particle (VLP) assembly assays:

  • Express ORF25 with or without other structural proteins to assess particle formation

  • Electron microscopy to visualize particle morphology

  • Gradient centrifugation to analyze particle size distribution and composition

  • Mutagenesis of ORF25 to identify regions critical for assembly

• Single-cycle infection analysis:

  • Create reporter viruses expressing tagged or fluorescent proteins

  • Time-course analysis with confocal microscopy to track ORF25 during infection

  • Correlative light and electron microscopy (CLEM) to connect protein localization with ultrastructural changes

  • Use of inhibitors at specific time points to dissect entry vs. assembly functions

• Membrane fusion and deformation assays:

  • Liposome-based fusion assays with fluorescent lipid mixing indicators

  • Giant unilamellar vesicle (GUV) deformation studies with purified ORF25

  • Atomic force microscopy to detect nanoscale membrane alterations

  • In vitro reconstitution of minimal assembly systems with purified components

• Host interaction identification during entry/assembly:

  • Proximity labeling at different time points during infection

  • Quantitative proteomics to identify time-dependent interaction changes

  • Live-cell imaging with split fluorescent proteins to visualize interactions during entry/assembly

  • Genetic screens to identify host factors specifically involved in ORF25-dependent steps

Similar methodological approaches applied to HEV proteins have revealed roles in viral egress via the exosomal pathway, providing a potential parallel for ORF25 function investigation . The temporal and spatial dynamics of ORF25 during infection can provide crucial insights into its role in the viral life cycle.

How should researchers interpret conflicting data regarding ORF25 function or localization?

When faced with conflicting data regarding ORF25 function or localization, a systematic approach to data interpretation and experimental design should be employed:

• Methodological reconciliation strategy:

  • Compare experimental conditions in detail (cell types, expression levels, tags used)

  • Evaluate temporal factors - observations at different time points may reflect dynamic processes

  • Consider tag interference effects - different tags or tag positions may affect protein behavior

  • Assess the sensitivity and specificity of detection methods used in conflicting studies

• Controlled comparative experiments:

  • Design experiments that directly test multiple conditions side-by-side

  • Include internal controls that can verify assay performance across experiments

  • Use orthogonal methods to address the same question (e.g., imaging plus biochemical fractionation)

  • Quantitative analysis with appropriate statistical methods to assess significance of differences

• Biological context considerations:

  • Evaluate whether conflicting data might reflect actual biological plasticity or context-dependence

  • Consider host cell factors that might influence protein behavior in different systems

  • Assess viral strain differences that might explain functional variations

  • Examine whether protein concentration effects could explain different observations

• Integrative data analysis:

  • Develop mathematical models that might accommodate seemingly conflicting observations

  • Use Bayesian approaches to weigh evidence based on methodological strengths

  • Consider ensemble behaviors rather than single states for membrane proteins

  • Employ machine learning to identify patterns across diverse datasets

This methodological framework has successfully resolved conflicts in viral protein function studies. For example, apparent contradictions in HEV ORF1 processing were addressed through careful methodological comparison and reconciliation of different experimental approaches .

What statistical approaches are most appropriate for analyzing quantitative data from ORF25 functional studies?

• Experimental design considerations:

  • Power analysis to determine appropriate sample sizes before conducting experiments

  • Randomization strategies to minimize batch effects and systematic errors

  • Blinding procedures for image analysis and phenotype scoring to reduce bias

  • Technical vs. biological replicates must be clearly distinguished in analysis

• Data preprocessing methodology:

  • Normalization approaches specific to the assay type (e.g., GAPDH for Western blots, housekeeping genes for qPCR)

  • Log transformation for data with multiplicative errors (common in biological systems)

  • Outlier detection using statistically rigorous methods (e.g., Grubbs' test, ROUT method)

  • Missing data handling that minimizes introduction of bias

• Statistical testing framework:

  • For normally distributed data: t-tests (paired/unpaired) or ANOVA with appropriate post-hoc tests

  • For non-parametric data: Mann-Whitney, Kruskal-Wallis with suitable post-hoc tests

  • For time-course experiments: repeated measures ANOVA or mixed-effects models

  • Multiple testing correction (Bonferroni, Benjamini-Hochberg) to control false discovery rate

• Advanced analytical approaches:

  • Multivariate analysis for complex datasets with multiple interdependent variables

  • Machine learning for pattern recognition in large-scale screening data

  • Bayesian inference for integrating prior knowledge with experimental results

  • Bootstrapping or permutation tests for datasets with unusual distributions

When applied to viral protein function studies, these approaches can reveal subtle but significant effects that might otherwise be missed. For example, statistical analysis of HEV replication efficiency in cells expressing mutant versions of viral proteins identified functional domains critical for viral replication .

What strategies can address poor expression or solubility of recombinant ORF25 protein?

Membrane proteins like ORF25 often present expression and solubility challenges that require systematic troubleshooting approaches:

• Expression system optimization:

  • Test multiple E. coli strains (BL21(DE3), C41(DE3), C43(DE3), Lemo21) specifically designed for membrane protein expression

  • Evaluate different expression vectors with varying promoter strengths

  • Optimize induction conditions (temperature reduction to 16-20°C, lower IPTG concentrations)

  • Consider alternative expression systems (insect cells, mammalian cells) for complex membrane proteins

• Fusion tag strategies:

  • N-terminal fusions with solubility enhancers (MBP, SUMO, TrxA)

  • Addition of purification tags at permissive sites identified through insertion screening

  • Inclusion of specific proteolytic cleavage sites for tag removal

  • C-terminal fusions when N-terminal elements are critical for folding

• Solubilization approach:

  • Detergent screening panel (start with DDM, LMNG, CHAPS, Fos-choline)

  • Detergent concentration optimization to minimize protein aggregation

  • Addition of lipids during solubilization to stabilize native conformation

  • Use of amphipathic polymers (amphipols, SMALPs) as alternatives to detergents

• Buffer optimization:

  • pH screening to identify stability optima

  • Salt concentration adjustment to enhance solubility

  • Addition of stabilizing agents (glycerol, trehalose, arginine)

  • Reducing agents (DTT, TCEP) to prevent disulfide-mediated aggregation

These strategies have been successfully applied to other viral membrane proteins, allowing structural and functional characterization despite initial solubility challenges. The methodical optimization of expression and purification conditions is essential for obtaining functional ORF25 protein for downstream analyses .

How can researchers overcome antibody detection limitations when studying ORF25?

Detection of viral proteins like ORF25 often presents challenges due to low expression levels or limited antibody sensitivity. A comprehensive strategy to overcome these limitations includes:

• Epitope tagging approaches:

  • Strategic insertion of small epitope tags (HA, FLAG, Myc) at permissive sites

  • Use of extensively validated commercial antibodies against these tags

  • Multi-tag strategies combining N-terminal and internal tags for signal amplification

  • Screening of multiple insertion sites to identify those that maintain protein function

• Custom antibody development optimization:

  • Careful antigen design focusing on predicted surface-exposed regions

  • Multiple rabbit immunization with different antigenic regions

  • Monoclonal antibody development for increased specificity

  • Extensive validation including knockout controls and heterologous expression systems

• Signal amplification methods:

  • Tyramide signal amplification for immunofluorescence

  • Proximity ligation assay (PLA) for enhanced sensitivity

  • Poly-HRP secondary antibodies for Western blot enhancement

  • Combination with fluorescence in situ hybridization (FISH) to detect protein-RNA complexes

• Alternative detection strategies:

  • Metabolic labeling with click chemistry-compatible amino acids

  • SNAP/CLIP-tag fusions for covalent labeling with synthetic fluorophores

  • Split complementation assays that generate signal only upon correct protein folding

  • Mass spectrometry-based detection of signature peptides

These approaches have successfully addressed detection challenges with other viral proteins. For example, the HEV ORF1
protein was notoriously difficult to detect until tagged constructs were developed . Similar strategies can be applied to overcome detection limitations for His1 virus ORF25.