Recombinant Invertebrate iridescent virus 6 Uncharacterized protein 234R (IIV6-234R)

What is the structural composition of IIV6-234R?

IIV6-234R is an uncharacterized protein from Invertebrate iridescent virus 6 (IIV-6), also known as Chilo iridescent virus. The recombinant form is typically expressed with a His-tag in E. coli expression systems. The full-length protein consists of 193 amino acids . The protein is encoded within the 234 non-overlapping open reading frames (ORFs) identified in the IIV-6 genome, which has a total size of 212 kbp with 28.6% G+C content . The function of this protein within the viral lifecycle remains incompletely characterized, making it an interesting target for structural and functional studies.

What expression systems are most effective for producing recombinant IIV6-234R?

Based on current research protocols, E. coli serves as the predominant expression system for recombinant IIV6-234R production . When expressing this protein, researchers typically use a His-tag to facilitate purification through affinity chromatography. The complete expression and purification workflow includes:

• Cloning the IIV6-234R coding sequence into an appropriate expression vector

• Transforming the construct into a compatible E. coli strain (typically BL21(DE3) or similar)

• Inducing protein expression under optimized conditions (temperature, induction time, IPTG concentration)

• Cell lysis and protein extraction

• Purification via Ni-NTA or similar affinity chromatography

• Secondary purification steps if higher purity is required

• Quality control assessment via SDS-PAGE and Western blotting

How does IIV6-234R contribute to the virus's ability to activate immune responses in mammalian cells?

While the specific contribution of IIV6-234R to immune activation has not been definitively characterized, IIV-6 as a whole has demonstrated the ability to stimulate type I interferon-dependent antiviral immune responses in mammalian cells . Interestingly, despite being a DNA virus, IIV-6 activates the RIG-I-like receptor (RLR) pathway typically associated with RNA virus detection, rather than the canonical cGAS/STING DNA sensing pathway . This activation occurs because RNA polymerase III transcribes viral DNA into RNA species capable of triggering the RLR pathway. The resulting immune response can protect mammalian cells from subsequent infection with arboviruses such as Vesicular Stomatitis virus and Kunjin virus . Whether IIV6-234R plays a direct role in this immune evasion or activation process represents an important research question.

What experimental approaches can determine if IIV6-234R interacts with specific components of the mammalian immune system?

To investigate potential interactions between IIV6-234R and mammalian immune components, researchers should consider a multi-faceted approach:

• Co-immunoprecipitation (Co-IP) studies:

  • Express tagged IIV6-234R in mammalian cells

  • Perform pull-down assays followed by mass spectrometry to identify interacting partners

  • Validate interactions by reciprocal Co-IP with candidate immune proteins

• Yeast two-hybrid screening:

  • Use IIV6-234R as bait against a mammalian immune component library

  • Validate positive interactions with alternative methods

• ELISA-based interaction studies:

  • Measure IFN-β secretion in response to purified IIV6-234R using established protocols

  • Compare responses between wild-type and immune component knockout cells

• Reporter assays:

  • Transfect cells with ISRE-firefly luciferase and TK-Renilla luciferase

  • Measure responses to IIV6-234R exposure using dual-luciferase assays

• Immunofluorescence microscopy:

  • Track cellular localization of IIV6-234R and potential immune components

  • Assess colocalization patterns during different stages of cellular response

What are the optimal conditions for studying IIV6-234R in vitro?

Based on established protocols for IIV-6 research, the following conditions are recommended for in vitro studies of IIV6-234R:

Cell Culture Conditions:

• For mammalian cell studies: 37°C/5% CO₂ in DMEM supplemented with 2% FBS

• For insect cell studies: 28°C in appropriate insect cell media

Infection/Transfection Protocols:

• For virus-based studies: Infect cells at an MOI of 1 TCID₅₀/cell for one hour at 28°C

• Wash cells three times with PBS post-infection

• Replace media with 2% FBS/DMEM and maintain at 37°C/5% CO₂

Protein Expression and Purification:

• Express recombinant IIV6-234R with a His-tag in E. coli systems

• Purify using affinity chromatography followed by size exclusion chromatography

• Store purified protein at -80°C in small aliquots to avoid freeze-thaw cycles

Analysis Methods:

• For protein-protein interactions: Co-IP, pull-down assays

• For functional assays: ELISA for cytokine production, reporter assays

• For structural studies: Circular dichroism, X-ray crystallography, or cryo-EM

What imaging techniques are most informative for studying IIV6-234R localization and function?

Several imaging approaches have proven valuable for iridovirus research and can be applied to IIV6-234R studies:

• Confocal Microscopy:

  • Fix cells at appropriate time points (e.g., 8 hours post-infection) with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton-X and block with 10% FBS in PBS

  • Use fluorescently labeled antibodies against IIV6-234R and cellular markers

  • Visualize nuclear translocation of transcription factors like NFκB using specific antibodies

• Transmission Electron Microscopy (TEM):

  • Fix cells in 2% paraformaldehyde/2% glutaraldehyde in 0.1M cacodylate buffer with 0.2M sucrose

  • Dehydrate in ethanol, infiltrate with Spurrs' resin, section, and stain with 4% uranyl acetate and Reynolds lead

  • Image using a transmission electron microscope to visualize viral particles and cellular ultrastructure

• Live-Cell Imaging:

  • Express fluorescently tagged IIV6-234R in living cells

  • Track protein movement and interactions in real-time

  • Combine with phase contrast microscopy to correlate with cellular morphological changes

• Super-Resolution Microscopy:

  • Apply techniques such as STORM or PALM for nanoscale resolution of IIV6-234R localization

  • Combine with proximity ligation assays to detect protein-protein interactions with high spatial precision

How can researchers effectively measure the immune response triggered by IIV6-234R?

To quantify immune responses to IIV6-234R, researchers should implement a comprehensive set of assays:

• Cytokine Production:

  • Collect supernatant from cells expressing or exposed to IIV6-234R

  • Measure IFN-β levels using ELISA according to manufacturer's protocols (e.g., PBL Assay Science 42410-1)

  • Assess production of other cytokines using multiplex assays

• Reporter Assays:

  • Transfect HEK 293T cells with ISRE-firefly luciferase and TK-Renilla luciferase constructs

  • Expose cells to supernatants from IIV6-234R-expressing cells

  • After incubation (e.g., 16 hours), lyse cells and measure dual luciferase activity

• Western Blotting for Signaling Pathway Activation:

  • Assess phosphorylation status of key proteins including:

    • IRF3 using anti-IRF3 (1:1,000) and anti-P-IRF3 (1:2,000)

    • STAT1 using anti-STAT1 (1:1,000) and anti-P-STAT (1:1,000)

    • IκB using anti-IκB (1:1,000) and anti-P-IκB (1:1,000)

• Protection Assays:

  • Pre-treat cells with purified IIV6-234R or control

  • Challenge with reporter viruses (e.g., VSV or KUNV)

  • Quantify protection by measuring viral replication or cell viability

How might IIV6-234R be utilized in developing novel antiviral strategies?

IIV6-234R's potential in antiviral research stems from IIV-6's unique ability to trigger protective immune responses against subsequent arboviral infections . Researchers could explore:

• Adjuvant Development:

  • Determine if purified IIV6-234R alone can stimulate protective immune responses

  • Test IIV6-234R as an adjuvant with existing vaccines to enhance immunogenicity

  • Develop modified versions with enhanced stability or immunostimulatory properties

• Broad-Spectrum Antiviral Strategies:

  • Investigate if pre-treatment with IIV6-234R protects against diverse viral families

  • Identify the minimum structural components needed for immune activation

  • Develop synthetic analogues based on IIV6-234R's immunostimulatory domains

• Mechanistic Studies:

  • Map the specific regions of IIV6-234R responsible for immune activation

  • Create targeted mutations to enhance or modify immunostimulatory properties

  • Investigate synergistic effects with other immunomodulatory molecules

• Delivery Systems:

  • Develop nanoparticle-based delivery of IIV6-234R to specific cell types

  • Test liposomal formulations for enhanced stability and cellular uptake

  • Explore nucleic acid-based expression systems for in vivo production

What are the most promising approaches for resolving the structure of IIV6-234R?

Determining the three-dimensional structure of IIV6-234R would significantly advance understanding of its function. The following approaches are recommended:

• X-ray Crystallography:

  • Express and purify large quantities of highly pure IIV6-234R

  • Screen various crystallization conditions systematically

  • Optimize promising conditions for diffracting crystals

  • Collect diffraction data and solve the structure

• Cryo-Electron Microscopy:

  • Particularly valuable if IIV6-234R forms complexes or if crystallization proves challenging

  • Apply single-particle analysis techniques

  • Consider using methods similar to those that produced 3D reconstructions of IIV-6 particles

• NMR Spectroscopy:

  • Suitable if IIV6-234R is relatively small or if focusing on specific domains

  • Requires isotope labeling (¹⁵N, ¹³C) during recombinant expression

  • Provides dynamic information not available from static methods

• Integrative Structural Biology:

  • Combine multiple techniques (SAXS, HDX-MS, crosslinking-MS)

  • Use computational modeling to generate structural predictions

  • Validate models with limited experimental data

• AlphaFold or Similar AI-Based Prediction:

  • Generate computational models as starting points for experimental validation

  • Compare predicted structures with homologous proteins of known function

  • Use predictions to guide mutagenesis studies

How can researchers investigate potential interactions between IIV6-234R and host proteins?

To comprehensively map IIV6-234R's interactome, researchers should implement the following complementary approaches:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express tagged IIV6-234R in relevant cell types

  • Perform pull-down experiments under various conditions

  • Identify interacting partners by mass spectrometry

  • Create interaction network maps from identified proteins

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse IIV6-234R to a biotin ligase (BirA*)

  • Express in cells to biotinylate proteins in close proximity

  • Purify biotinylated proteins and identify by mass spectrometry

  • Provides information on spatial relationships in living cells

• Yeast Two-Hybrid Screening:

  • Use IIV6-234R as bait against cDNA libraries from relevant host species

  • Validate interactions through secondary screens

  • Test direct binding with purified proteins

• Protein Microarrays:

  • Probe arrays containing purified host proteins with labeled IIV6-234R

  • Identify binding partners through fluorescence or other detection methods

  • Quantify relative binding affinities

• Surface Plasmon Resonance (SPR):

  • Measure binding kinetics between IIV6-234R and candidate interacting proteins

  • Determine association and dissociation rates

  • Calculate binding affinities

How does IIV6-234R compare to homologous proteins in other iridoviruses?

To understand the evolutionary context of IIV6-234R, researchers should conduct comprehensive comparative analyses:

• Sequence Comparison:

  • Perform BLAST searches against other iridovirus genomes

  • Identify proteins with sequence similarity in related viruses

  • Create multiple sequence alignments to identify conserved domains

  • Note that no collinearity has been observed between the genomes of IIV-3 and IIV-6 , suggesting significant evolutionary divergence

• Structural Prediction Comparison:

  • Generate structural models of IIV6-234R and homologs

  • Compare predicted structural features across viral species

  • Identify conserved structural motifs that may indicate functional importance

• Functional Domain Analysis:

  • Identify conserved functional domains using databases like Pfam or PROSITE

  • Compare domain architecture across homologous proteins

  • Correlate domain presence with known functional differences

• Evolutionary Rate Analysis:

  • Calculate selective pressure (dN/dS) across protein sequences

  • Identify regions under positive or purifying selection

  • Correlate evolutionary rates with structural features

What can we learn from comparing infection mechanisms of IIV-6 across different host species?

IIV-6 demonstrates a remarkably broad host range, especially when administered through injection . Researchers investigating host-pathogen interactions should:

• Compare Infection Efficiency:

  • Quantify infection rates across insect orders and other arthropods

  • Investigate why injection routes show broader host range than oral routes

  • Determine if IIV6-234R expression levels correlate with successful infection

• Host Response Comparison:

  • Compare immune responses to IIV-6 infection across host species

  • Identify conserved and divergent defense mechanisms

  • Determine if IIV6-234R interacts differently with immune components across species

• Cell Entry Mechanisms:

  • Investigate whether IIV6-234R plays a role in cell entry or tissue tropism

  • Compare entry efficiency across cell types from different host species

  • Develop fluorescently labeled virus particles to track early infection events

• Cross-Species Transmission Factors:

  • Identify viral and host factors that enable IIV-6's broad host range

  • Determine whether IIV6-234R contributes to cross-species infection potential

  • Compare with more host-restricted iridoviruses like IIV-3, IIV-16, or IIV-24