Recombinant Invertebrate iridescent virus 3 Uncharacterized protein 124R (IIV3-124R)

How is the recombinant version of IIV3-124R typically produced?

Recombinant IIV3-124R protein is typically expressed using E. coli expression systems. The production process involves cloning the coding sequence into an expression vector that incorporates an N-terminal histidine tag (His-tag) for purification purposes . For genomic DNA extraction, IIV-3 infected mosquito larvae are first ground with a Tekmar Tissuemizer in deionized water and filtered through a 400 mesh to remove large insect parts. The filtrate is then layered on a continuous HS-40 Ludox gradient and centrifuged for isolation . For recombinant protein expression, the DNA is typically digested with appropriate restriction enzymes, cloned into plasmid vectors (such as pUC19), and grown in Escherichia coli . The resulting protein is purified using affinity chromatography techniques that take advantage of the His-tag.

What are the physicochemical properties of the IIV3-124R protein?

Based on its amino acid sequence and typical storage conditions, the IIV3-124R protein has the following properties:

The protein contains regions with repeated sequences, particularly "LAVSPLAVSPLAVSPL" which appears as a triplet repeat within the sequence .

What are the optimal conditions for expressing recombinant IIV3-124R in E. coli?

For optimal expression of recombinant IIV3-124R in E. coli, researchers should consider the following parameters:

• Vector selection: Use expression vectors with strong promoters such as T7 or tac promoters.

• E. coli strain optimization: BL21(DE3) or Rosetta strains are recommended for efficient expression of viral proteins.

• Induction conditions: Typically, IPTG concentrations of 0.5-1.0 mM at OD600 of 0.6-0.8 yield good expression.

• Temperature considerations: Lowering the induction temperature to 18-25°C may increase the solubility of the expressed protein.

• Expression time: Optimal expression is usually achieved after 4-6 hours of induction at 37°C or overnight induction at lower temperatures.

After harvesting the cells, lysis can be performed using sonication or pressure-based homogenization in a buffer containing protease inhibitors to prevent degradation of the recombinant protein. Purification can then be achieved using Ni-NTA affinity chromatography, taking advantage of the His-tag .

What purification methods are most effective for recombinant IIV3-124R?

The most effective purification strategy for recombinant His-tagged IIV3-124R typically involves a multi-step approach:

• Initial capture using IMAC (Immobilized Metal Affinity Chromatography):

  • Use Ni-NTA or Co-NTA resins with a binding buffer containing 20-50 mM imidazole

  • Elute with an imidazole gradient (100-250 mM)

• Secondary purification:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Ion exchange chromatography based on the protein's theoretical pI

• Buffer optimization:

  • Final formulation in Tris-based buffer with 50% glycerol for stability

  • pH adjustment to maintain protein stability (typically pH 7.5-8.0)

Purification should be performed at 4°C to minimize protein degradation, and the final product should be aliquoted to avoid repeated freeze-thaw cycles, which can significantly impact protein integrity .

How can researchers verify the identity and purity of recombinant IIV3-124R?

Verification of recombinant IIV3-124R identity and purity should include multiple analytical techniques:

• SDS-PAGE analysis: To assess protein purity and approximate molecular weight

• Western blotting: Using anti-His antibodies to confirm the presence of the tagged protein

• Mass spectrometry:

  • Peptide mass fingerprinting for protein identification

  • Intact mass analysis to confirm the full-length protein

• N-terminal sequencing: To verify the correct start of the protein

• Dynamic light scattering: To assess homogeneity and detect aggregation

• Circular dichroism: To evaluate secondary structure

Researchers should aim for >90% purity as determined by SDS-PAGE analysis for most functional studies .

What is known about the function of IIV3-124R in viral infection and replication?

The function of IIV3-124R in the viral life cycle remains largely uncharacterized, which presents significant research opportunities. Based on studies of related iridoviruses, potential functions may include:

• Structural component: It may serve as a capsid or envelope protein contributing to virion structure.

• Host interaction: The protein may be involved in host cell recognition, attachment, or entry.

• Immune evasion: It could potentially play a role in modulating host immune responses.

• Replication: The protein might participate in viral genome replication or assembly.

Understanding the function of IIV3-124R could provide insights into the virus's pathogenicity and host range. Comparative analysis with other iridovirus proteins may offer clues to its role, particularly given that some iridescent viruses like IIV6 have demonstrated insecticidal activities in various pest species including Lepidoptera and Coleoptera .

How does IIV3-124R compare with similar proteins in other invertebrate iridescent viruses?

Comparative analysis of IIV3-124R with homologous proteins in other invertebrate iridescent viruses reveals interesting evolutionary patterns:

Research focusing on comparative genomics and proteomics between IIV-3 and IIV-6 is particularly valuable, as IIV-6 has been shown to infect a broader range of pest insect species and has been explored for potential biopesticide applications. Recombinant IIV-6 variants incorporating insect toxin genes (such as the scorpion toxin AaIT) have demonstrated enhanced insecticidal activities against pests including Helicoverpa armigera, Lymantria dispar, and Tenebrio molitor .

What approaches can be used to study protein-protein interactions involving IIV3-124R?

To investigate potential protein-protein interactions involving IIV3-124R, researchers can employ multiple complementary techniques:

• Yeast two-hybrid screening:

  • Construct bait plasmids containing IIV3-124R fused to DNA-binding domains

  • Screen against prey libraries from relevant host cells

• Co-immunoprecipitation:

  • Use anti-His antibodies to pull down recombinant IIV3-124R

  • Identify interacting partners by mass spectrometry

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proximal proteins in relevant cell types

  • TurboID for rapid labeling of interacting proteins

• Surface plasmon resonance (SPR):

  • Immobilize purified IIV3-124R on sensor chips

  • Measure binding kinetics with potential partner proteins

• Protein crosslinking mass spectrometry:

  • Identify interaction interfaces at amino acid resolution

  • Map structural relationships between protein complexes

These methods can help elucidate the role of IIV3-124R in viral replication and host interactions, potentially revealing new targets for antiviral intervention or biotechnological applications.

What cell-based assays are most appropriate for studying IIV3-124R function?

For functional characterization of IIV3-124R, researchers should consider these cell-based assays:

• Infection models:

  • Mosquito cell lines (C6/36, Aag2) for native host studies

  • Other insect cell lines to assess host range

• Localization studies:

  • Fluorescent protein fusions (GFP-IIV3-124R) for live-cell imaging

  • Immunofluorescence with anti-His or custom antibodies in fixed cells

• Overexpression and knockdown studies:

  • Transient expression to assess cytopathic effects

  • CRISPR interference for genetic knockdown during viral infection

• Binding assays:

  • Flow cytometry to measure cell surface binding

  • Cell fractionation to determine subcellular localization

• Reporter systems:

  • Luciferase-based assays to monitor effects on host gene expression

  • Split reporter systems to visualize protein-protein interactions

Selecting appropriate cell lines that support IIV-3 replication is crucial for meaningful functional studies. The recombinant protein approach can complement virus infection studies to dissect specific protein functions.

How can structural studies enhance our understanding of IIV3-124R?

Structural characterization of IIV3-124R would significantly advance understanding of its function through:

• X-ray crystallography:

  • High-resolution structural determination requires:

    • Protein at >95% purity

    • Concentrations of 5-15 mg/mL

    • Screening numerous crystallization conditions

• Cryo-electron microscopy:

  • Single-particle analysis for structure determination

  • In situ localization within virions

• NMR spectroscopy:

  • For dynamic regions and intrinsically disordered segments

  • Requires isotope-labeled protein expression (15N, 13C)

• Small-angle X-ray scattering (SAXS):

  • Low-resolution envelope determination

  • Analysis of conformational states in solution

• Computational approaches:

  • AlphaFold2 or RoseTTAFold predictions

  • Molecular dynamics simulations to study flexibility

These structural insights would facilitate structure-based hypotheses about IIV3-124R function and potentially reveal similarities to proteins of known function, despite limited sequence homology.

What potential biotechnological applications exist for recombinant IIV3-124R?

While IIV3-124R remains functionally uncharacterized, several potential biotechnological applications can be explored:

• Biopesticide development:

  • Assessment of insecticidal activities similar to studies with recombinant IIV6

  • Potential fusion with insect-specific toxins like the scorpion toxin AaIT, which has shown effectiveness in recombinant IIV6 constructs against pests including Helicoverpa armigera and Lymantria dispar

• Diagnostic applications:

  • Development of antibodies against IIV3-124R for virus detection

  • ELISA-based diagnostic kits for environmental monitoring

• Vector control strategies:

  • Exploration of IIV3-124R as a mosquito control agent

  • Assessment of effects on vector competence in disease transmission

• Protein engineering platforms:

  • Use as a scaffold for presenting antigens or functional domains

  • Design of chimeric proteins with enhanced stability or functionality

Research exploring these applications would benefit from comparative studies with better-characterized iridescent viruses like IIV6, which has demonstrated potential as a biological control agent when modified with recombinant DNA technology .

What are common challenges in working with recombinant IIV3-124R and how can they be addressed?

Researchers often encounter several technical challenges when working with recombinant IIV3-124R:

For optimal results, recombinant IIV3-124R should be stored at -20°C/-80°C with 5-50% glycerol as a cryoprotectant. Working aliquots can be kept at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they significantly reduce protein integrity .

How can researchers validate antibodies against IIV3-124R for experimental use?

Proper validation of antibodies against IIV3-124R requires a systematic approach:

• Initial validation:

  • Western blot analysis using recombinant protein as a positive control

  • Testing against lysates from IIV-3-infected cells vs. uninfected controls

• Specificity testing:

  • Pre-absorption with recombinant protein to demonstrate specificity

  • Testing against related iridescent virus proteins to assess cross-reactivity

• Application-specific validation:

  • For immunofluorescence: co-localization studies with tagged recombinant protein

  • For immunoprecipitation: recovery efficiency using spiked recombinant protein

  • For ELISA: standard curves using purified recombinant protein

• Knockout/knockdown controls:

  • Genetic knockdown systems to verify signal specificity

  • Heterologous expression systems as positive controls

Commercial antibodies should be assessed using at least three independent validation methods before use in critical experiments.

What considerations are important when designing experiments to study IIV3-124R in the context of insect cells?

When studying IIV3-124R in insect cell systems, researchers should address these key considerations:

• Cell line selection:

  • Natural host cells (Ochlerotatus taeniorhynchus-derived) for physiological relevance

  • Commonly used insect cell lines (Sf9, High Five, S2) for experimental tractability

• Expression systems:

  • Baculovirus expression for high-level protein production

  • Inducible systems to control expression timing and levels

• Physiological conditions:

  • Temperature optimization (20-28°C range for most insect cells)

  • Appropriate media formulations for specific insect cell types

• Viral infection controls:

  • Comparison with other iridescent virus proteins (e.g., from IIV6)

  • Time-course studies to capture dynamic processes

• Functional readouts:

  • Cytopathic effect monitoring

  • Host gene expression changes

  • Cell cycle and apoptosis measurements

Experiments should include appropriate positive controls (known IIV proteins) and negative controls (unrelated viral or cellular proteins) to ensure result specificity and interpretability.

What genomic and proteomic approaches could advance our understanding of IIV3-124R?

Several cutting-edge approaches could significantly advance IIV3-124R research:

• Comparative genomics:

  • Pan-genome analysis of IIV-3 isolates from different geographic regions

  • Evolutionary analysis of 124R homologs across the Iridoviridae family

• Transcriptomics:

  • RNA-seq analysis of host responses to recombinant IIV3-124R

  • Temporal expression profiling during infection

• Proteomics:

  • Interactome mapping using proximity labeling followed by mass spectrometry

  • Post-translational modification profiling

• Functional genomics:

  • CRISPR screens to identify host factors interacting with IIV3-124R

  • Transposon mutagenesis to map essential domains

• Single-cell approaches:

  • Single-cell RNA-seq to characterize heterogeneity in host responses

  • Mass cytometry for protein-level single-cell analysis

These approaches could be integrated with datasets like FluPRINT, which demonstrates how multi-dimensional biological measurements, including mass cytometry combined with multiple high-dimensional biological measurements, can reveal heterogeneity of immune system responses .

How might recombinant IIV3-124R be modified to enhance its utility in research and applications?

Strategic modifications to IIV3-124R could enhance its research utility:

• Fusion partners:

  • Fluorescent proteins (GFP, mCherry) for localization studies

  • Affinity tags beyond His-tag (FLAG, GST) for alternative purification strategies

  • Split reporter systems for interaction studies

• Domain engineering:

  • Truncation constructs to identify functional domains

  • Chimeric proteins with homologous regions from other iridescent viruses

• Stability enhancements:

  • Surface entropy reduction for crystallization

  • Disulfide engineering for increased thermostability

• Functionality additions:

  • Addition of cell-penetrating peptides for cellular delivery

  • Incorporation of insect-specific toxins similar to successful approaches with IIV6

• Expression optimization:

  • Codon optimization for specific expression systems

  • Removal of cryptic splice sites or regulatory elements

Successful engineering approaches could draw inspiration from recombinant IIV6 constructs that have incorporated green fluorescent protein (GFP) or scorpion toxins to create viruses with enhanced features for biological control applications .

What interdisciplinary approaches could accelerate discoveries related to IIV3-124R?

Progress in IIV3-124R research could be accelerated through interdisciplinary collaborations:

• Structural biology and computational approaches:

  • Integration of experimental structural data with AI prediction tools

  • Molecular dynamics simulations to predict functional sites

• Vector biology and epidemiology:

  • Assessment of IIV-3 prevalence in wild mosquito populations

  • Evaluation of effects on vector competence for disease transmission

• Agricultural and environmental sciences:

  • Field studies of modified IIV3-124R constructs for pest management

  • Environmental impact assessments

• Systems biology:

  • Network analysis to position IIV3-124R in host-pathogen interaction networks

  • Multi-omics integration for comprehensive functional characterization

• Synthetic biology:

  • Design of synthetic variants with engineered functions

  • Development of genetic circuits incorporating IIV3-124R regulatory elements

These interdisciplinary approaches could help bridge gaps between basic research on IIV3-124R and potential applications in vector control, agriculture, or biotechnology.