Recombinant His1 virus Putative transmembrane protein ORF32 (ORF32)

What is His1 virus and where does ORF32 fit within its genome?

His1 virus (Haloarcula hispanica virus 1) is a lemon-shaped haloarchaeal virus that infects archaea in extreme salt environments. It belongs to a distinctive group of archaeal viruses with unusual morphology. ORF32 is one of the putative transmembrane proteins encoded in the His1 virus genome, with potential roles in viral structure and host interaction . The virus exhibits a unique lemon-shaped capsid with a tail structure that plays a key role in host recognition and infection processes. The genome of His1 virus contains multiple ORFs encoding structural and functional proteins, with ORF32 being part of the membrane-associated protein repertoire.

How does His1 virus differ from other archaeal viruses in structure and function?

His1 virus exhibits a distinctive lemon-shaped morphology with a uniform tail structure that distinguishes it from other archaeal viruses. Unlike many icosahedral or rod-shaped viruses, His1 has a pleomorphic capsid that can transform into tubular structures under certain conditions . Cryo-electron tomography studies have revealed that while the His1 capsid shows size and shape heterogeneity, its tail structure remains constant, featuring a central hub with six tail spikes that likely facilitate host attachment . This morphological adaptability may represent an evolutionary advantage for survival in extreme environments. Unlike tailed bacteriophages, His1 is non-lytic, suggesting it uses different mechanisms for virion assembly and release from host cells .

What are the optimal conditions for recombinant expression of ORF32 in E. coli?

For optimal expression of recombinant ORF32 in E. coli, researchers should consider the following protocol:

• Vector selection: Use expression vectors containing strong promoters (T7 or tac) with an N-terminal His-tag for purification.

• Host strain: BL21(DE3) or Rosetta strains are recommended for membrane proteins.

• Growth conditions: Culture at 30°C rather than 37°C to reduce inclusion body formation.

• Induction parameters: Use 0.1-0.5 mM IPTG at OD600 0.6-0.8, then continue expression at 18-20°C overnight.

• Media supplementation: Add 1% glucose to suppress basal expression and 5-10% glycerol to stabilize the protein.

This approach has been demonstrated to yield functional recombinant protein suitable for structural and functional studies .

What purification strategies are most effective for obtaining high-purity ORF32 protein?

A multi-step purification protocol is recommended for obtaining high-purity ORF32:

• Cell lysis: Use detergent-based lysis buffer (e.g., 1% Triton X-100 or n-dodecyl β-D-maltoside) with protease inhibitors.

• Initial capture: Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with imidazole gradient elution (20-250 mM).

• Intermediate purification: Apply size exclusion chromatography using Superdex 75 or 200 columns in buffer containing 0.05-0.1% detergent.

• Final polishing: If necessary, use ion exchange chromatography to remove remaining contaminants.

• Quality assessment: Verify purity by SDS-PAGE (should show > 90% purity) and Western blotting.

This protocol typically yields 2-5 mg of purified protein per liter of bacterial culture .

How can researchers validate the correct folding and functionality of purified ORF32?

Validating correct folding and functionality of purified ORF32 requires multiple complementary approaches:

• Circular dichroism (CD) spectroscopy: Analyze secondary structure content and thermal stability.

• Fluorescence spectroscopy: Assess tertiary structure integrity through intrinsic tryptophan fluorescence.

• Size-exclusion chromatography with multi-angle light scattering (SEC-MALS): Determine oligomeric state and homogeneity.

• Lipid bilayer integration assay: Confirm membrane insertion capacity using liposome flotation assays.

• Functional binding assays: Test interaction with potential host cell components or other viral proteins.

Researchers should look for consistent spectroscopic profiles across different purification batches and stability in detergent micelles or reconstituted membrane environments.

What membrane topology does ORF32 adopt and how does this relate to its function?

ORF32 likely adopts a multi-pass transmembrane topology with hydrophobic segments spanning the membrane multiple times. Computational topology predictions suggest:

• The N-terminal region (residues 1-25) is cytoplasmic, containing charged residues.

• Three to four transmembrane helices span residues 26-120.

• The C-terminal domain (residues 121-143) faces the exterior and may interact with other viral or host components.

This topology is functionally significant as it positions different protein domains on opposite sides of the membrane, enabling ORF32 to potentially participate in:

• Viral envelope formation and structure

• Host cell membrane recognition

• Potential fusion events during viral entry

• Signal transduction across viral membranes

The predicted transmembrane arrangement shares characteristics with other viral membrane proteins involved in host-virus interactions .

How does ORF32 contribute to the unusual lemon-shaped morphology of His1 virus?

While direct structural evidence is limited, several mechanisms suggest how ORF32 might contribute to His1's distinctive morphology:

• Membrane curvature induction: ORF32's transmembrane domains likely contain wedge-shaped segments that induce membrane curvature necessary for the lemon-shaped capsid.

• Protein-protein interactions: ORF32 may form specific oligomeric assemblies that create scaffolding for the virus shape, similar to how viral proteins in other systems define virion morphology.

• Capsid-membrane interface: ORF32 could serve as an adaptor between the capsid proteins and lipid envelope, creating tension that maintains the characteristic lemon shape.

• Structural transformations: Research has shown that under certain conditions, the His1 viral capsid can transform into tube-like structures . ORF32 might be involved in regulating these conformational changes, which are likely important during host infection.

Cryo-electron tomography studies have revealed that while His1 exhibits size and shape heterogeneity, its tail structure remains constant , suggesting ORF32 might particularly influence the capsid structure rather than the tail components.

What protein-protein interactions does ORF32 participate in during viral assembly?

ORF32 likely engages in multiple protein-protein interactions during viral assembly:

• Homotypic interactions: ORF32 proteins may oligomerize to form structural elements within the viral envelope. These interactions could involve specific motifs in the transmembrane regions.

• Capsid protein interactions: ORF32 likely interacts with major capsid proteins that form the lemon-shaped body. This interaction may involve the C-terminal domain of ORF32, which is predicted to be exposed to the viral interior.

• Tail protein associations: Given the importance of the tail structure for host attachment, ORF32 may interact with proteins that form the tail complex, potentially contributing to the organization of the central hub and six tail spikes observed in cryo-electron tomography studies .

• Genome packaging interactions: Some transmembrane viral proteins interact with nucleic acids or nucleoproteins during genome packaging. ORF32 might similarly participate in organizing the viral genome within the capsid.

Further biochemical and structural studies using techniques like co-immunoprecipitation, crosslinking mass spectrometry, or proximity labeling would be valuable to fully map these interaction networks.

What methods are recommended for studying ORF32 integration into artificial membrane systems?

For studying ORF32 integration into artificial membrane systems, researchers should consider these methodological approaches:

• Liposome reconstitution:

  • Prepare liposomes with lipid compositions mimicking archaeal membranes (e.g., bipolar tetraether lipids)

  • Use detergent-mediated reconstitution followed by dialysis or detergent adsorption

  • Confirm integration via sucrose gradient flotation assays

• Nanodiscs preparation:

  • Utilize archaeal lipid-compatible membrane scaffold proteins

  • Optimize lipid-to-protein and scaffold-to-ORF32 ratios

  • Characterize by size-exclusion chromatography and electron microscopy

• Planar lipid bilayers:

  • Form bilayers across apertures in Teflon partitions

  • Monitor conductance changes upon ORF32 addition

  • Assess voltage-dependent behaviors

• Giant unilamellar vesicles (GUVs):

  • Prepare GUVs containing fluorescently labeled lipids

  • Add fluorescently labeled ORF32

  • Visualize using confocal microscopy to assess membrane deformation

Each approach provides complementary information about membrane integration, structural perturbations, and potential functional properties of ORF32 in membrane environments.

How can researchers assess the impact of mutations in ORF32 on viral infectivity?

A comprehensive approach to assess the impact of mutations in ORF32 on viral infectivity includes:

• Rational mutation design:

  • Target conserved residues identified through sequence alignment

  • Focus on transmembrane helices and putative functional domains

  • Create alanine scanning libraries or targeted substitutions

• Recombinant virus generation:

  • Utilize bacterial artificial chromosome (BAC) systems for virus engineering

  • Create ORF32 mutant viruses through homologous recombination

  • Verify genome integrity through sequencing

• Infectivity assays:

  • Quantify viral entry using labeled virions

  • Measure viral replication by qPCR of viral genomes

  • Determine viral yield through plaque assays or TCID50

• Mechanistic studies:

  • Assess membrane fusion capacity using lipid mixing assays

  • Evaluate virion morphology by electron microscopy

  • Track intracellular trafficking of mutant viruses

• Complementation analysis:

  • Express wild-type ORF32 in trans to rescue mutant phenotypes

  • Create chimeric ORF32 proteins to map functional domains

This approach has been successfully applied to study functional domains in viral transmembrane proteins of other viruses and could be adapted for His1 ORF32 .

What imaging techniques are most informative for visualizing ORF32 localization in infected cells?

For optimal visualization of ORF32 localization in infected cells, researchers should employ these complementary imaging techniques:

• Immunofluorescence microscopy:

  • Generate specific antibodies against ORF32 or use epitope-tagged versions

  • Co-stain with markers for cellular compartments (ER, Golgi, plasma membrane)

  • Perform time-course studies to track localization changes during infection

  • Resolution: ~200 nm

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence imaging of ORF32 with ultrastructural context

  • Use gold-conjugated antibodies for EM detection

  • Identify ORF32 in the context of assembling virions

  • Resolution: 2-5 nm for EM component

• Super-resolution microscopy:

  • Apply STED, PALM, or STORM techniques

  • Achieve localization precision of 20-50 nm

  • Track single molecules of fluorescently labeled ORF32

  • Perform multi-color imaging with viral and cellular markers

• Cryo-electron tomography:

  • Visualize infected cells in near-native state

  • Locate ORF32 using immunogold labeling

  • Generate 3D reconstructions of virus-cell interactions

  • Resolution: 3-5 nm

• Live-cell imaging:

  • Create fluorescent protein fusions with ORF32

  • Monitor dynamic localization during infection

  • Measure protein mobility using FRAP or FCS

  • Assess interactions using FRET

These approaches have been successfully applied to study archaeal virus-host interactions and could provide valuable insights into ORF32 function .

How can ORF32 be incorporated into virus-like particles for potential biotechnology applications?

Incorporation of ORF32 into virus-like particles (VLPs) for biotechnology applications can be achieved through these methodological approaches:

• Co-expression systems:

  • Design constructs expressing ORF32 alongside major capsid proteins

  • Optimize expression ratios to ensure proper incorporation

  • Use insect cell or archaeal expression systems for authentic post-translational modifications

• Self-assembly protocols:

  • Purify individual components separately

  • Establish controlled in vitro assembly conditions

  • Monitor assembly using dynamic light scattering and electron microscopy

• Surface modification strategies:

  • Engineer ORF32 fusion proteins with functional domains (fluorescent proteins, targeting ligands)

  • Confirm surface exposure using antibody accessibility assays

  • Assess functional activity of the displayed domains

• Cargo encapsulation methods:

  • Develop disassembly/reassembly protocols in the presence of cargo molecules

  • Optimize buffer conditions to maintain particle integrity

  • Quantify loading efficiency using fluorescence or radioactive labeling

• Stability enhancement:

  • Introduce crosslinking sites for increased VLP stability

  • Test thermal and pH resistance of modified particles

  • Evaluate storage conditions for long-term preservation

This strategy builds upon successful approaches used with other archaeal virus proteins and leverages the stability of proteins evolved for extreme environments .

What are the challenges and solutions for studying protein-protein interactions between ORF32 and host cell receptors?

Studying protein-protein interactions between ORF32 and host cell receptors presents several challenges with corresponding methodological solutions:

These approaches have been adapted from successful strategies used to study virus-receptor interactions in other systems .

How does ORF32 compare structurally and functionally to equivalent proteins in other archaeal viruses?

ORF32 shares structural and functional features with transmembrane proteins from other archaeal viruses, though with important distinctions:

• Structural comparisons:

  • Like haloarchaeal tailed virus proteins, ORF32 contains hydrophobic transmembrane domains, but lacks the conserved portal-Mu gpF fusion domains found in viruses like HHTV-2 and HCTV-2

  • Unlike the major capsid proteins of spindle-shaped archaeal viruses such as Sulfolobus monocaudavirus 1 (SMV1), ORF32 does not appear to undergo the dramatic conformational changes that enable transformation from helical to quasi-spherical arrangements

  • ORF32's predicted membrane topology is similar to that of transmembrane proteins in other lemon-shaped viruses, suggesting conserved mechanisms for membrane interaction

• Functional parallels:

  • Similar to tail proteins in other archaeal viruses, ORF32 likely contributes to the stable tail structure of His1, which features a central hub with six tail spikes that maintain consistent organization despite capsid heterogeneity

  • The protein may function analogously to membrane proteins in other archaeal viruses that facilitate host recognition and attachment in extreme environments

  • Unlike the Orf virus (ORFV) vector systems that have been engineered to express heterologous proteins for vaccine development , His1 ORF32 has not yet been extensively explored for such applications

• Evolutionary implications:

  • Sequence analysis places ORF32 within the broader context of archaeal virus transmembrane proteins, though with limited sequence homology to characterized proteins

  • The protein represents part of the adaptive strategy of His1 virus to its extreme halophilic environment, similar to membrane adaptations seen in other archaeal viruses

  • Recent studies suggest spindle-shaped archaeal viruses evolved from rod-shaped predecessors , and transmembrane proteins like ORF32 may preserve evidence of this evolutionary trajectory

This comparative analysis provides context for understanding ORF32's role within the broader landscape of archaeal virus biology .

What methodological approaches can be used to study the role of ORF32 in His1 virus genome release during infection?

To investigate ORF32's role in His1 virus genome release, researchers should employ these methodological approaches:

• Real-time fluorescence microscopy:

  • Label His1 viral DNA with fluorescent nucleic acid dyes (SYBR Gold, PicoGreen)

  • Track genome release into host cells using time-lapse imaging

  • Compare wild-type virus with ORF32 mutants or in the presence of anti-ORF32 antibodies

• In vitro genome release assays:

  • Develop conditions that trigger DNA release from purified virions

  • Test effects of pH, temperature, and ion concentrations

  • Measure DNA release using fluorescence or gel electrophoresis

  • Compare virions with modified ORF32 variants

• Structural transitions analysis:

  • Leverage the observation that His1 capsids can transform into tubes during genome release

  • Monitor structural transitions using negative-stain EM or cryo-EM

  • Correlate structural changes with genome release events

  • Assess how ORF32 mutations affect these transformations

• Cross-linking mass spectrometry:

  • Apply time-resolved cross-linking during infection

  • Identify dynamic changes in ORF32 interactions

  • Map structural rearrangements associated with genome delivery

• Single-particle tracking:

  • Label ORF32 and viral DNA with different fluorophores

  • Track their relative movements during infection

  • Quantify the spatiotemporal relationship between ORF32 reorganization and genome release

This integrated approach would elucidate ORF32's specific role in the remarkable capsid-to-tube transition observed during His1 infection and genome delivery .

How might ORF32 be engineered as a delivery vehicle for archaeal genetic modification systems?

Engineering ORF32 as a delivery vehicle for archaeal genetic modification could be approached through:

• Domain engineering:

  • Identify minimal functional domains required for membrane integration

  • Create fusion proteins with DNA-binding domains for cargo capture

  • Design chimeric constructs combining ORF32 with cell-penetrating peptides

• Vesicle delivery system:

  • Incorporate ORF32 into archaeal-compatible liposomes

  • Optimize protein density for efficient host cell recognition

  • Package CRISPR-Cas components or expression constructs as cargo

• Virus-like particle adaptations:

  • Co-express modified ORF32 with compatible capsid proteins

  • Engineer assembly-competent particles with altered tropism

  • Incorporate cargo DNA through packaging signals or direct conjugation

• Targeting modifications:

  • Identify host-recognition domains within ORF32

  • Create variant libraries with altered species specificity

  • Screen for broader or narrower host range variants

• Entry mechanism optimization:

  • Characterize natural entry pathways mediated by ORF32

  • Enhance fusion capability through targeted mutations

  • Incorporate pH-responsive elements for controlled release

This engineering approach draws parallels to successful viral vector systems like recombinant herpesviruses, which have been engineered to express heterologous proteins for targeted delivery .

What insights might comparative studies of ORF32 across different archaeal virus families provide?

Comparative studies of ORF32 across archaeal virus families could yield several significant insights:

• Evolutionary relationships:

  • Trace the phylogenetic history of transmembrane proteins in archaeal viruses

  • Identify conserved domains that represent ancestral functions

  • Map diversification patterns that correlate with host adaptation

  • Test the hypothesis that spindle-shaped viruses evolved from rod-shaped predecessors

• Structure-function correlations:

  • Compare membrane topology predictions across virus families

  • Identify conserved motifs associated with specific functions

  • Correlate structural features with host range and environmental adaptations

• Host interaction mechanisms:

  • Compare receptor recognition domains across diverse archaeal viruses

  • Identify convergent strategies for host cell attachment

  • Map co-evolutionary patterns between viral proteins and host receptors

• Environmental adaptations:

  • Analyze how transmembrane proteins adapt to extreme conditions

  • Compare halophilic, thermophilic, and acidophilic adaptations

  • Identify amino acid compositions that confer stability in different environments

• Biotechnological applications:

  • Identify domains with unique properties for protein engineering

  • Discover stable scaffolds for membrane protein design

  • Develop archaeal virus-based tools optimized for extreme conditions

This comparative approach would place ORF32 in a broader evolutionary context and potentially reveal fundamental principles of virus-host interactions in archaea .

What are the methodological considerations for studying ORF32 interactions with archaeal lipid membranes?

Studying ORF32 interactions with archaeal lipid membranes requires specialized methodological considerations:

• Lipid composition challenges:

  • Archaeal membranes contain unique ether-linked lipids rather than ester-linked phospholipids

  • Commercial availability of archaeal lipids is limited

  • Solution: Extract lipids directly from archaeal cultures or use synthetic archaeol and caldarchaeol lipids

• Membrane fluidity differences:

  • Archaeal membranes have distinct fluidity properties compared to bacterial or eukaryotic membranes

  • Temperature and salt concentration critically affect membrane behavior

  • Solution: Maintain experimental conditions that mimic natural hypersaline environments (3-5M NaCl, pH 7-8)

• Reconstitution protocols:

  • Standard proteoliposome formation protocols may not work optimally with archaeal lipids

  • Solution: Modify detergent removal methods and lipid-to-protein ratios specifically for archaeal systems

  • Validate membrane integration using sucrose gradient flotation assays

• Analytical techniques:

  • Fluorescence approaches may be affected by high salt conditions

  • Solution: Use salt-resistant fluorophores and correct for solvent effects

  • Employ multiple complementary techniques (FRET, EPR, ATR-FTIR) for robust measurements

• Functional assays:

  • Traditional vesicle permeability assays may be complicated by archaeal membrane properties

  • Solution: Develop archaeal-specific permeability markers and standardized assay conditions

  • Include appropriate controls with known archaeal membrane proteins