Recombinant His1 virus Putative transmembrane protein ORF24 (ORF24)

What is the His1 virus and why is its ORF24 protein of interest to researchers?

His1 is a novel archaeal virus isolated from hypersaline waters in southeastern Australia. It infects Haloarcula hispanica, displaying a distinctive lemon-shaped morphology (74 by 44 nm) with a short tail, and belongs to the Fuselloviridae family of archaeal viruses . ORF24 is of particular interest as it represents one of the few characterized transmembrane proteins from archaeal viruses, potentially offering insights into viral-host interactions in extreme environments.

The His1 virus has several notable features that make its proteins worthy of study:

• It has a linear double-stranded DNA genome of 14.9 kb, which is the smallest recorded for any halophilic virus

• It exhibits remarkable resistance to low salt concentrations despite infecting halophilic archaea

• It establishes a persistent carrier state with its host rather than forming lysogens

• It represents the first halophilic member of the Fuselloviridae family

What expression systems are used to produce recombinant ORF24 protein?

Recombinant ORF24 is typically expressed in E. coli systems with a His-tag to facilitate purification . The commonly used approach includes:

• Cloning the full-length ORF24 gene (1-88 amino acids) into a bacterial expression vector

• Transformation into an E. coli strain optimized for protein expression

• Induction of expression using IPTG or similar inducers

• Purification using affinity chromatography (typically Ni-NTA for His-tagged proteins)

• Storage in a Tris-based buffer with 50% glycerol for stability

What are the optimal conditions for maintaining the stability of purified recombinant ORF24?

Based on established protocols for similar viral transmembrane proteins, the following conditions are recommended for maintaining ORF24 stability:

Researchers should avoid repeated freeze-thaw cycles as this can lead to protein degradation and loss of structural integrity . For experiments requiring buffer exchange, consider gradual dialysis to maintain protein solubility and prevent aggregation.

What techniques are most effective for studying the membrane insertion and topology of ORF24?

Several complementary techniques can be employed to study ORF24's membrane topology:

• Fluorescence Microscopy with GFP Fusion Constructs: Creating N-terminal and C-terminal GFP fusion proteins can help determine the orientation of ORF24 in the membrane. This approach has been successfully used with other viral transmembrane proteins to visualize localization and topology .

• Protease Protection Assays: By exposing membrane-embedded ORF24 to proteases and then analyzing the protected fragments by mass spectrometry, researchers can identify which regions are embedded in the membrane versus exposed to the solvent.

• Cysteine Scanning Mutagenesis: Systematically replacing individual amino acids with cysteine and then using membrane-impermeable sulfhydryl reagents to determine which residues are accessible from which side of the membrane.

• Cryo-Electron Microscopy: For higher-resolution structural analysis, cryo-EM has proven effective for examining transmembrane proteins, as demonstrated with designed transmembrane pores . Though challenging with smaller proteins like ORF24, this approach could provide valuable structural insights.

How can researchers assess the oligomerization state of ORF24 in membranes?

Determining the oligomerization state of ORF24 requires several complementary approaches:

• Size Exclusion Chromatography (SEC): SEC can provide initial insights into whether ORF24 exists as monomers, dimers, tetramers, or higher-order oligomers in detergent solutions.

• Chemical Cross-linking: Using membrane-permeable cross-linking agents followed by SDS-PAGE analysis can capture transient protein-protein interactions.

• Förster Resonance Energy Transfer (FRET): By labeling different populations of ORF24 with donor and acceptor fluorophores, FRET can detect close proximity between protein molecules, indicating oligomerization.

• Analytical Ultracentrifugation: This technique can determine the molecular weight of protein-detergent complexes in solution, helping to ascertain oligomeric states.

• Native Mass Spectrometry: This emerging technique can measure the intact mass of membrane protein complexes, revealing the precise oligomeric state and stoichiometry.

In recent studies, transmembrane proteins have shown diverse oligomerization states from monomers to tetramers, with the oligomeric state often correlating with function .

What methodologies can be used to investigate ORF24's role in His1 virus infection?

To elucidate ORF24's role in viral infection, researchers should consider a multi-pronged approach:

• Gene Knockout or Mutation Studies: Though challenging in archaeal viruses, CRISPR-based approaches or recombineering techniques could be adapted to create ORF24 mutants. Comparing the infectivity of wild-type and mutant viruses would provide insights into ORF24's importance.

• Co-immunoprecipitation with Host Proteins: Using antibodies against tagged ORF24 to pull down associated host proteins could identify potential interaction partners.

• Localization Studies: Fluorescence microscopy using labeled antibodies against ORF24 could track its distribution during different stages of infection.

• Membrane Permeability Assays: If ORF24 forms pores, as some viral transmembrane proteins do, membrane permeability assays using fluorescent dyes could detect changes in membrane integrity during infection.

• Heterologous Expression in Host Cells: Expressing ORF24 alone in Haloarcula hispanica could reveal whether it has cytotoxic effects independent of other viral proteins.

Studies with other archaeal viruses suggest transmembrane proteins can mediate various functions including viral entry, release, or modification of host metabolism .

How does ORF24 compare to transmembrane proteins from other archaeal viruses?

Comparative analysis reveals several key distinctions and similarities:

Unlike larger and more complex archaeal viruses such as HHIV-2, which contains at least 15 different structural proteins divided into vertex, capsid, and membrane-associated categories , His1's ORF24 represents a simpler transmembrane system. This simplicity makes ORF24 a valuable model for understanding fundamental aspects of virus-host membrane interactions in archaeal systems.

What is known about the potential role of ORF24 in viral assembly or host cell entry?

While specific experimental data on ORF24's role is limited, inferences can be made based on similar viral transmembrane proteins:

• Viral Assembly Hypothesis: ORF24 could function during viral assembly by facilitating the incorporation of viral membrane components derived from host cells. This role would be consistent with observations in other enveloped viruses where transmembrane proteins act as anchors for capsid components.

• Host Entry Mechanism: Alternatively, ORF24 might participate in host recognition or membrane fusion processes. The persistent carrier state established by His1 suggests a complex interaction with the host membrane that could involve ORF24.

• Membrane Remodeling: ORF24 might alter host cell membrane properties to facilitate viral replication or release without cell lysis, consistent with His1's ability to exit without immediately causing cell lysis .

To test these hypotheses, researchers could employ site-directed mutagenesis targeting key residues in ORF24, followed by functional assays measuring virus production and host cell interactions.

How can computational modeling be applied to predict ORF24 structure and function?

Computational approaches offer powerful tools for studying ORF24:

• Homology Modeling: Though challenging due to limited homologs with known structures, advances in protein structure prediction tools like AlphaFold or RoseTTAFold could generate reasonably accurate models of ORF24.

• Molecular Dynamics Simulations: Once a structural model is established, MD simulations can reveal how ORF24 behaves within a lipid bilayer, including potential conformational changes and lipid interactions.

• Coevolutionary Analysis: Methods like direct coupling analysis (DCA) can identify potentially interacting residues within ORF24 or between ORF24 and host proteins based on evolutionary constraints.

• Energy Function-Based Design: Approaches similar to those used in de novo transmembrane protein design can help predict optimal membrane insertion and stability of ORF24 variants.

Recent advances in computational design of transmembrane proteins demonstrate that accurate structural predictions are increasingly feasible, even for proteins with complex membrane topologies .

How can researchers design experiments to determine if ORF24 forms homooligomeric structures?

To investigate potential homooligomerization of ORF24, researchers should consider the following experimental design:

• In vitro Reconstitution Studies:

  • Express and purify recombinant ORF24 with and without fusion tags

  • Reconstitute the protein in various membrane mimetics (nanodiscs, liposomes, detergent micelles)

  • Analyze oligomeric state using techniques like SEC-MALS (size exclusion chromatography with multi-angle light scattering)

• Genetic Approaches:

  • Create fusion constructs with split reporter proteins (e.g., split GFP)

  • Co-express both halves in cells - signal will only occur if proteins interact

  • Quantify interaction strength through fluorescence measurements

• Computational Analysis of Interfaces:

  • Predict potential oligomerization interfaces using computational methods similar to those used in designing transmembrane oligomers

  • Validate predictions through targeted mutagenesis of interfacial residues

Recent studies have shown that transmembrane proteins can form stable, defined oligomeric structures including dimers, trimers, and tetramers through specifically designed interfaces , suggesting similar potential for naturally occurring proteins like ORF24.

What techniques can be employed to study ORF24 interactions with host membrane components?

To investigate ORF24-host membrane interactions, researchers can use these methodologies:

• Lipid Binding Assays:

  • Create liposomes with different lipid compositions reflecting host membrane

  • Measure ORF24 binding affinity to different lipid compositions using fluorescence techniques

  • Determine specific lipid preferences that might be important for function

• Surface Plasmon Resonance (SPR):

  • Immobilize ORF24 or potential host interaction partners on SPR chips

  • Measure real-time binding kinetics and affinities

  • Test effects of different buffer conditions, including salt concentration relevant to halophilic environments

• Proximity Labeling:

  • Express ORF24 fused to enzymes like BioID or APEX in host cells

  • These enzymes will biotinylate proteins in close proximity to ORF24

  • Identify labeled proteins by mass spectrometry to map the ORF24 interactome

• Native Mass Spectrometry:

  • Extract membrane sections containing ORF24 under native conditions

  • Analyze by native MS to identify co-purifying lipids or proteins

  • Determine specific host factors that interact with ORF24

Similar approaches have proven effective in studying virus-host interactions in other systems and could provide valuable insights into ORF24's role in His1 infection of Haloarcula hispanica.

How does ORF24 compare to transmembrane proteins from other extremophilic viruses?

ORF24 from His1 virus provides an interesting comparison with transmembrane proteins from other extremophilic viruses:

The comparison between ORF24 and transmembrane proteins from other extremophilic viruses reveals important adaptations to extreme environments. Unlike thermophilic viral proteins that must resist heat denaturation, ORF24 likely contains adaptations for functioning in hypersaline environments, such as an abundance of acidic residues on exposed surfaces and unique folding patterns .

The fact that His1 virus particles are resistant to low salt concentrations suggests that ORF24 may incorporate structural features that maintain stability across varying salt conditions—a valuable property for potential biotechnological applications.

What evolutionary insights can be gained from studying ORF24 and related viral transmembrane proteins?

Studying ORF24 in an evolutionary context provides several important insights:

• Domain Evolution: Analysis of ORF24 can help understand how transmembrane domains evolved in archaeal viruses, which represent some of the most ancient viral lineages.

• Host Adaptation: Comparing ORF24 with transmembrane proteins from viruses infecting different hosts can reveal adaptations specific to halophilic archaea versus other hosts.

• Functional Conservation: Identifying conserved motifs between ORF24 and other viral transmembrane proteins might reveal functionally important regions preserved through evolution.

• Horizontal Gene Transfer: Analyzing whether ORF24 shows similarity to host proteins could indicate potential horizontal gene transfer events in the evolution of His1 virus.

The lemon-shaped morphology of His1 virus appears to be an archaeal-specific trait not found among bacterial or eukaryotic viruses , suggesting this viral morphotype has a deep evolutionary history within the archaeal domain. ORF24, as a component of this system, represents an opportunity to study protein evolution in one of life's most ancient viral lineages.

How can researchers use recombinant ORF24 as a tool for understanding archaeal virus-host interactions?

Recombinant ORF24 offers several applications for investigating archaeal virus-host dynamics:

• Binding Studies: Purified ORF24 can be used to identify potential receptors or binding partners on the host cell surface through direct binding assays.

• Competition Assays: Pre-incubating host cells with recombinant ORF24 before viral infection can determine if it competes with infectious virions for cellular receptors.

• Structural Comparative Analysis: The structure of ORF24 can be compared with host membrane proteins to identify similarities that might explain host range specificity.

• Lipid Interaction Profiling: Determining which host membrane lipids preferentially interact with ORF24 could provide insights into viral tropism and membrane fusion mechanisms.

• Immunological Tools: Antibodies raised against recombinant ORF24 can be used to study the distribution and dynamics of the protein during infection.

The persistent carrier state established by His1 virus , rather than the lysogenic state seen with other temperate viruses, represents a unique virus-host relationship. Understanding how ORF24 contributes to this persistent relationship could reveal novel mechanisms of viral persistence relevant to understanding virus-host coevolution.

What emerging technologies might advance our understanding of ORF24 structure and function?

Several cutting-edge technologies show promise for elucidating ORF24 properties:

• Cryo-Electron Tomography: This technique could visualize ORF24 in its native membrane environment within intact virions, providing structural insights in context.

• Integrative Structural Biology: Combining multiple structural techniques (X-ray crystallography, NMR, cryo-EM, computational modeling) could overcome the challenges of determining membrane protein structures.

• Single-Molecule Techniques: Methods like single-molecule FRET or optical tweezers could reveal dynamic aspects of ORF24 function that are inaccessible to bulk measurements.

• Nanopore Technology: Adapting techniques from transmembrane pore research could allow functional characterization of ion or small molecule transport through ORF24, if it forms pores.

• Advanced Computational Methods: Machine learning approaches for protein structure prediction and molecular dynamics simulations with specialized force fields for membrane proteins could provide more accurate models of ORF24.

Recent advances in de novo design of transmembrane proteins with specific oligomerization states and functions demonstrate the feasibility of accurately modeling and predicting the behavior of transmembrane proteins like ORF24.

How might ORF24 be adapted as a research tool for membrane biology studies?

ORF24's unique properties offer several applications as a research tool:

• Membrane Protein Fusion Partner: ORF24's stability in different salt conditions could make it a useful fusion partner for expressing and studying other challenging membrane proteins.

• Halophilic Membrane Model System: As a relatively simple transmembrane protein from an extremophile, ORF24 provides a model system for studying membrane protein adaptation to extreme environments.

• Archaeal-Specific Cell Surface Targeting: If ORF24's binding partners on archaeal cells are identified, it could be used to target molecules specifically to archaeal cell surfaces in mixed microbial communities.

• Minimal Membrane Anchor: The compact size of ORF24 makes it a candidate for use as a minimal membrane anchor in synthetic biology applications requiring membrane localization.