Recombinant Invertebrate iridescent virus 6 Uncharacterized protein 135R (IIV6-135R)

What is Invertebrate iridescent virus 6 and what are its key genomic features?

Invertebrate iridescent virus 6 (IIV-6), also known as Chilo iridescent virus, is a large double-stranded DNA virus belonging to the family Iridoviridae, specifically within the genus Iridovirus of the subfamily Betairidovirinae. The virus features a substantial genome of 212,482 base pairs with 215 non-overlapping open reading frames (ORFs) . The genome has a relatively low G+C content of 28.63%, which is characteristic of insect-infecting iridoviruses within this genus .

IIV-6 has a complex genomic organization with ORFs distributed on both strands of the viral genome—approximately 45% on the upper (R) strand and 55% on the lower (L) strand . This bidirectional coding strategy indicates complex transcriptional regulation and suggests that convergent overlapping transcription likely generates double-stranded RNA during infection, which can trigger host immune responses .

How are IIV-6 uncharacterized proteins classified within the viral genome?

Uncharacterized proteins in IIV-6, including protein 135R, are named according to their position in the genome and the strand from which they are transcribed. The number represents the open reading frame's position, while the letter (R or L) indicates the genomic strand—R for the "upper" strand read from left to right, and L for the "lower" complementary strand .

These uncharacterized proteins are identified through bioinformatic analysis of the viral genome but lack experimental validation of their functions. While the IIV-6 genome contains 215 non-overlapping ORFs, the functions of many encoded proteins remain unknown, presenting significant challenges and opportunities for researchers . Based on genomic position and transcriptional direction, researchers can develop hypotheses about potential functional relationships with neighboring genes or other characterized viral proteins.

What is the structural organization of IIV-6 virions and how might this relate to uncharacterized proteins?

IIV-6 virions exhibit a complex trilaminar structure consisting of three concentric domains: an outer proteinaceous capsid, an intermediate lipid membrane with associated polypeptides, and an inner nucleocytoplasmic dsDNA genome core . The icosahedral capsid follows a triangulation number of T=147 lattice, contributing to the virus's structural stability and large size.

The virion's diameter measures approximately 120-130 nm in ultrathin sections, but can reach up to 185 nm when including the fibrils emanating from the capsid surface . Uncharacterized proteins like 135R may be components of any of these structural domains, potentially serving roles in capsid integrity, membrane association, or nucleic acid interactions. Understanding the localization of these proteins within the virion structure is crucial for determining their functional roles in the viral life cycle.

What are the recommended protocols for expressing and purifying recombinant IIV-6 proteins for functional studies?

The efficient expression and purification of recombinant IIV-6 proteins like 135R typically involves a multi-stage process optimized for viral proteins. Based on established protocols for related IIV-6 proteins:

• Expression system selection: Mammalian cell expression systems are often preferred for viral proteins requiring eukaryotic post-translational modifications . For IIV-6 proteins, researchers should consider using human embryonic kidney (HEK293) or Chinese hamster ovary (CHO) cell lines for expression.

• Vector design: The coding sequence should be codon-optimized for the selected expression system and cloned into an appropriate vector containing a strong promoter (such as CMV). Incorporate purification tags (His, FLAG, or GST) positioned to minimize interference with protein folding and function .

• Purification protocol: For recombinant IIV-6 proteins, a typical purification workflow includes:

  • Cell lysis under conditions that maintain protein solubility

  • Initial capture using affinity chromatography based on the fusion tag

  • Secondary purification via ion exchange or size exclusion chromatography

  • Quality assessment by SDS-PAGE to verify >85% purity, similar to other IIV-6 recombinant proteins

• Storage optimization: For maximum stability, recombinant viral proteins should be stored with 5-50% glycerol at -20°C/-80°C to prevent freeze-thaw damage. Liquid formulations typically maintain stability for 6 months, while lyophilized preparations can remain stable for up to 12 months .

How can researchers effectively design experiments to investigate IIV-6 infection dynamics?

When designing experiments to study IIV-6 infection, researchers should implement comprehensive experimental designs that account for multiple variables and potential confounders:

• Experimental group design: Implement true experimental designs with proper control groups. For IIV-6 studies, this often requires:

  • Experimental group receiving IIV-6 infection

  • Mock-infected control group receiving vehicle only

  • Positive control group with a characterized virus (if comparing viral effects)

• Randomization and replication: Ensure random assignment of subjects to experimental and control groups to minimize selection bias. For insect models, this requires standardizing age, size, and developmental stage .

• Infection protocol standardization: Establish consistent viral dose (known as multiplicity of infection or MOI), inoculation route, and time points for analysis. For Drosophila models, intraabdominal inoculation has been effectively used for IIV-6 .

• Measurement protocols: Implement multi-parameter assessment including:

  • Viral titer quantification over time

  • Visual markers of infection (e.g., iridescence in infected tissues)

  • Molecular markers such as viral protein detection via Western blot

  • Host response measurements (transcriptomic, proteomic, or functional assays)

• Timeline considerations: Monitor infection progression over appropriate timescales. Research indicates IIV-6 can establish productive infection in model organisms with significant viral titer increases within 6 days post-infection .

What methodological approaches can determine the subcellular localization and binding partners of IIV6-135R?

To comprehensively characterize the subcellular localization and interaction network of IIV6-135R, researchers should employ complementary approaches:

• Fluorescence microscopy techniques:

  • Generate fluorescent protein fusions (GFP-135R) for live-cell imaging

  • Perform immunofluorescence with antibodies against the tagged protein

  • Implement co-localization studies with organelle markers

  • Consider super-resolution microscopy for detailed subcellular distribution

• Biochemical fractionation:

  • Separate infected cells into nuclear, cytoplasmic, membrane, and organelle fractions

  • Analyze fractions by Western blotting to determine protein distribution

  • Complement with density gradient ultracentrifugation to isolate viral assembly intermediates

• Protein-protein interaction identification:

  • Conduct co-immunoprecipitation (Co-IP) using antibodies against the tagged protein

  • Implement proximity-based labeling methods (BioID or APEX) to identify neighboring proteins

  • Apply yeast two-hybrid or mammalian two-hybrid screening to identify direct interactions

  • Validate interactions using recombinant proteins in vitro with techniques like surface plasmon resonance

• Cross-linking mass spectrometry:

  • Apply in vivo crosslinking to capture transient interactions

  • Perform liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify crosslinked peptides

  • Map interaction interfaces through computational analysis of crosslinked residues

The integration of these complementary approaches provides comprehensive insight into both localization patterns and the interaction network of IIV6-135R, establishing a foundation for functional characterization.

How does the host RNA interference pathway interact with IIV-6 infection and what implications does this have for uncharacterized protein research?

The interplay between IIV-6 and the host RNA interference (RNAi) system represents a critical aspect of host-pathogen interaction with important implications for studying uncharacterized proteins:

Research demonstrates that IIV-6, despite being a DNA virus, triggers the production of Dicer-2 (Dcr-2)-dependent viral small interfering RNAs (vsiRNAs) in infected hosts . This activation of RNAi occurs because IIV-6's bidirectional transcription from both genomic strands generates double-stranded RNA intermediates, which are recognized and processed by the host RNAi machinery .

For researchers investigating uncharacterized proteins like IIV6-135R, this RNAi interaction presents several methodological considerations:

• Experimental approach for RNAi analysis:

  • Sequence small RNAs (19-30 nucleotides) from infected hosts using next-generation sequencing

  • Map small RNAs to the viral genome to identify hotspots of vsiRNA production

  • Compare vsiRNA profiles between wild-type hosts and RNAi-deficient mutants (e.g., Dcr-2 mutants)

  • Correlate vsiRNA abundance with transcript levels of specific viral genes

• Functional implications:

  • Proteins targeted by abundant vsiRNAs may be particularly important for viral replication

  • Temporal analysis of vsiRNA production can reveal expression kinetics of uncharacterized proteins

  • Uncharacterized proteins might function in RNAi suppression or evasion

• Research applications:

  • Use RNAi-based knockdown to assess the function of uncharacterized proteins

  • Develop RNAi escape mutants to identify essential viral genes

  • Compare vsiRNA profiles across different host species to identify conserved targets

The integration of RNAi pathway analysis with traditional protein characterization approaches provides a powerful strategy for elucidating the functions of uncharacterized viral proteins in the context of host-pathogen interactions.

What bioinformatic approaches are most effective for predicting the structure and function of uncharacterized IIV-6 proteins?

For uncharacterized proteins like IIV6-135R, applying a systematic bioinformatic pipeline can substantially enhance functional predictions:

• Sequence-based analysis:

  • Implement position-specific iterated BLAST (PSI-BLAST) to identify remote homologs

  • Perform multiple sequence alignment with other iridovirus proteins to identify conserved motifs

  • Apply hidden Markov model (HMM) profiling to detect functional domains

  • Utilize specialized viral protein databases like pVOGs (prokaryotic Virus Orthologous Groups)

• Structural prediction approaches:

  • Generate tertiary structure predictions using AlphaFold2 or RoseTTAFold

  • Validate structural models through molecular dynamics simulations

  • Perform structural alignment against known protein structures to identify potential functional analogs

  • Map conserved residues onto the predicted structure to identify functional sites

• Integrated functional prediction:

  • Analyze predicted post-translational modifications using NetPhos, NetOGlyc, and similar tools

  • Implement subcellular localization prediction using DeepLoc or PSORT

  • Apply computational tools specifically designed for viral proteins like VIRALpro

  • Utilize gene neighborhood analysis to identify functional associations with adjacent genes

• Evolutionary analysis:

  • Construct phylogenetic trees to determine evolutionary relationships with characterized proteins

  • Calculate selection pressure (dN/dS ratios) to identify regions under positive selection

  • Compare synteny across related viral genomes to identify conserved gene clusters

  • Apply coevolution analysis to predict protein-protein interactions

For uncharacterized IIV-6 proteins, these bioinformatic approaches should be applied within the context of the known genomic characteristics (28.63% G+C content) and taxonomic relationships (within the oligoiridovirus group) , enhancing the specificity and accuracy of the predictions.

How can comparative genomics between different IIV isolates inform functional predictions for uncharacterized proteins?

Comparative genomic analysis across different iridovirus isolates provides powerful insights into the potential functions of uncharacterized proteins like IIV6-135R:

• Conservation analysis across iridovirus species:
  The Iridoviridae family contains multiple genera with varying genomic characteristics as illustrated in the table below:

  Genus	Representative Species	Genome Size (bp)	ORF Count	G+C Content	Host Range
  Iridovirus	IIV-6	212,482	215	28.63%	Insects, arthropods
  Iridovirus	IIV-31	220,222	203	35.09%	Crustaceans
  Chloriridovirus	IIV-3	191,132	126	48%	Diptera (mosquitoes)
  Chloriridovirus	IIV-9	205,791	191	31%	Diptera

  By analyzing the presence, absence, and sequence similarity of the 135R ortholog across these diverse iridoviruses, researchers can infer:

  • Evolutionary age of the protein (widespread conservation suggests ancient origin)

  • Host-specific adaptations (conservation in specific host-adapted viral lineages)

  • Functional importance (highly conserved proteins typically serve essential functions)

• Synteny analysis:

  • Examine the genomic context of 135R across different iridovirus genomes

  • Identify consistently co-occurring genes that may function in the same pathway

  • Detect operon-like structures indicating coordinated expression

  • Look for evidence of horizontal gene transfer events

• Sequence divergence patterns:

  • Calculate nucleotide and amino acid substitution rates across orthologous sequences

  • Identify conserved motifs and domains that may be functionally important

  • Analyze selective pressure (dN/dS ratios) to detect regions under positive or purifying selection

  • Examine insertion/deletion patterns that may relate to host adaptation

• Application to functional prediction:

  • Proteins conserved across all iridoviruses likely serve core viral functions

  • Proteins unique to specific genera may relate to host range or tissue tropism

  • Rapidly evolving regions might indicate immune evasion functions

  • Conservation patterns can guide targeted mutagenesis for functional validation

This comparative approach has been successful in resolving ambiguous relationships between iridovirus isolates and improving classification , demonstrating its utility for functional prediction of uncharacterized proteins.

What are the main challenges in expressing and purifying IIV-6 proteins, and what strategies can overcome these limitations?

Researchers face several significant challenges when working with recombinant IIV-6 proteins:

• Solubility limitations:
  IIV-6 structural proteins often exhibit poor solubility due to their hydrophobic properties and tendency to aggregate. To address this:

  • Implement solubility-enhancing fusion tags (MBP, SUMO, or TRX)

  • Optimize expression temperature (typically lowering to 16-18°C)

  • Screen multiple detergents for membrane-associated viral proteins

  • Consider coexpression with viral or host chaperone proteins

• Protein stability issues:
  Many viral proteins show limited stability outside their native environment. Strategies to improve stability include:

  • Add stabilizing agents like glycerol (5-50%) to storage buffers

  • Determine optimal pH and ionic strength through thermal shift assays

  • Implement flash-freezing protocols to avoid freeze-thaw damage

  • Consider lyophilization for long-term storage (improving shelf life from 6 to 12 months)

• Post-translational modifications:
  Insect virus proteins may require specific post-translational modifications for proper folding and function:

  • Select expression systems capable of appropriate modifications (mammalian cells preferred)

  • Verify modification status using mass spectrometry

  • Consider insect cell expression systems to better recapitulate native modifications

  • Implement site-directed mutagenesis to eliminate problematic modification sites

• Functional validation:
  For uncharacterized proteins like 135R, confirming proper folding is challenging:

  • Develop functional assays based on predicted protein activities

  • Implement circular dichroism to assess secondary structure integrity

  • Use thermal denaturation assays to compare stability with native protein

  • Validate interaction partners as a proxy for correct folding

Each recombinant IIV-6 protein requires empirical optimization of these parameters, with careful documentation of successful conditions to establish reproducible protocols.

How can researchers design control groups appropriately when studying IIV-6 protein function?

• Control design hierarchy:
  The experimental design should include multiple levels of controls:

  • Negative controls (mock treatments, vehicle only)

  • Positive controls (well-characterized viral proteins with known functions)

  • Internal controls (housekeeping genes/proteins unaffected by viral infection)

  • System controls (testing in multiple cell types or host organisms)

• Controls for recombinant protein studies:
  When studying specific IIV-6 proteins like 135R:

  • Express and purify a non-relevant protein using identical methods

  • Create inactive mutants through site-directed mutagenesis of predicted active sites

  • Generate truncated protein variants lacking specific domains

  • Use unrelated viral proteins from the same structural class

• Addressing threats to internal validity:
  Experimental designs must control for several types of validity threats :

  • Non-comparable groups: Use randomization and matching techniques

  • Endogenous change: Include time-matched controls

  • External events: Conduct parallel control experiments

  • Contamination: Implement rigorous sterility controls

  • Treatment misidentification: Verify protein identity through mass spectrometry

• Specialized controls for RNA interference studies:
  When examining IIV-6 interactions with RNAi pathways:

  • Include RNAi-deficient host models (e.g., Dcr-2 mutants)

  • Use non-targeting siRNAs as controls

  • Compare effects across multiple viral genes

  • Consider host species with varying RNAi capacities

A properly designed control strategy addresses the "reactivity" problem described in experimental design literature , ensuring that observed effects are due to the viral protein's function rather than artifacts of the experimental system.

What strategies can resolve contradictory results in IIV-6 protein characterization studies?

When faced with contradictory results in viral protein characterization studies, researchers should implement a systematic resolution approach:

• Methodological reconciliation:

  • Conduct detailed comparison of experimental protocols between contradictory studies

  • Identify potential variables that might explain differences (cell types, protein tags, buffers)

  • Implement standardized protocols across laboratories to minimize technical variation

  • Consider interlaboratory validation studies with identical reagents and protocols

• Biological context exploration:

  • Investigate how different host systems might affect protein function

  • Examine temporal dynamics throughout infection (early vs. late expression)

  • Consider viral strain variations that might affect protein structure or function

  • Evaluate the influence of host factors and cellular conditions

• Integrated multi-approach validation:

  • Combine orthogonal techniques to verify findings (e.g., structural, biochemical, and genetic approaches)

  • Implement CRISPR/Cas9 genome editing of viral genomes to validate protein function

  • Apply mathematical modeling to reconcile seemingly contradictory observations

  • Develop quantitative rather than qualitative readouts to better compare results

• Data integration framework:

  • Implement meta-analysis methodologies to systematically evaluate contradictory results

  • Develop weighted evidence schemes based on methodological rigor

  • Consider Bayesian approaches to update confidence in specific hypotheses as new data emerges

  • Establish community standards for minimal information reporting in iridovirus studies

This systematic approach acknowledges that contradictions often reveal important biological complexities rather than experimental failures, potentially leading to deeper understanding of context-dependent protein functions.

What emerging technologies hold the most promise for characterizing IIV-6 uncharacterized proteins?

Several cutting-edge technologies are poised to revolutionize our understanding of uncharacterized viral proteins like IIV6-135R:

• Cryo-electron microscopy (cryo-EM) and tomography:

  • Single-particle cryo-EM can reveal the structure of purified 135R at near-atomic resolution

  • Cryo-electron tomography can localize 135R within the complex IIV-6 virion structure

  • Correlative light and electron microscopy (CLEM) can track 135R during the viral life cycle

  • Focused ion beam milling combined with tomography can visualize 135R in the cellular context

• High-throughput functional genomics:

  • CRISPR interference/activation screens in insect cells can identify host factors interacting with 135R

  • Massively parallel reporter assays can characterize regulatory functions

  • Global thermal proteome profiling can identify drug targets and binding partners

  • Synthetic genomics approaches to create minimal viable IIV-6 variants can determine essentiality

• Advanced structural prediction and modeling:

  • AI-driven structure prediction (AlphaFold2, RoseTTAFold) can generate highly accurate structural models

  • Molecular dynamics simulations can predict protein dynamics and functional states

  • Integrative modeling combining sparse experimental data with computational prediction

  • Deep mutational scanning to map sequence-function relationships

• Single-virus and single-cell technologies:

  • Single-virus tracking to monitor the dynamics of fluorescently labeled 135R during infection

  • Single-cell transcriptomics to characterize cell-to-cell variation in response to 135R expression

  • Single-cell proteomics to identify changes in the host proteome upon viral infection

  • Microfluidics-based assays to study infection dynamics at the single-cell level

These technologies, especially when applied in combination, offer unprecedented resolution for characterizing previously inaccessible aspects of viral protein function within the complex host-pathogen interface.

How might uncharacterized IIV-6 proteins contribute to the development of biotechnological tools?

Uncharacterized viral proteins like IIV6-135R represent untapped resources for biotechnological innovation:

• Novel enzyme discovery:

  • DNA/RNA modifying enzymes with unique properties

  • Protein modification catalysts with potential applications in protein engineering

  • Metabolic enzymes with industrial applications

  • Polymerases with distinctive fidelity or substrate preferences

• Cell biology research tools:

  • Protein translocation or trafficking regulators for manipulating cellular compartments

  • Novel fluorescent protein scaffolds for imaging applications

  • Regulators of host cell gene expression for synthetic biology

  • Viral capsid proteins as nanocontainers for drug delivery

• Immunological applications:

  • Novel adjuvants or immune modulators

  • Protein scaffolds for vaccine development

  • Immune evasion mechanisms with therapeutic potential

  • Diagnostic reagents for detecting insect viruses

• Synthetic biology components:

  • Genetic parts for orthogonal regulatory systems

  • Novel DNA-binding domains for synthetic transcription factors

  • Protein-protein interaction domains for building synthetic signaling pathways

  • Membrane-modifying proteins for creating artificial vesicles or organelles

The unique evolutionary history of IIV-6 as an insect-specific virus suggests its proteins may have distinctive properties optimized for functioning in arthropod hosts, potentially offering novel activities not found in better-characterized mammalian virus systems or bacterial proteins currently used in biotechnology.