Recombinant Invertebrate iridescent virus 6 Putative myristoylated membrane protein 458R (IIV6-458R)

What is Invertebrate iridescent virus 6 and the role of its myristoylated membrane protein 458R?

Invertebrate iridescent virus 6 (IIV-6), also known as Chilo iridescent virus, is a large, icosahedral, double-stranded DNA virus belonging to the family Iridoviridae, genus Iridovirus . The virus infects a wide range of invertebrate hosts but has also been detected in poikilothermic vertebrates including reptiles and amphibians .

The putative myristoylated membrane protein 458R (IIV6-458R) is encoded by the IIV6-458R gene and is classified among core IIV genes involved in virion formation . This protein is characterized by:

• Function: Contributes to viral membrane structure and possibly host cell interactions

• Classification: Core virion formation protein

• UniProt accession number: Q91F68

• Expression region: 2-495

• Sequence length: Full protein length is 494 amino acids

The protein's myristoylation suggests it plays a role in membrane anchoring, potentially facilitating virus assembly or host cell membrane interactions during viral entry or egress.

What are the optimal storage and handling conditions for recombinant IIV6-458R?

For optimal stability and activity of recombinant IIV6-458R, the following storage and handling conditions are recommended:

The high glycerol content (50%) in the storage buffer is critical for maintaining the integrity of the membrane protein structure. Additionally, the protein's myristoylation modification must be preserved for structural studies, requiring careful temperature management .

What expression systems are most effective for producing recombinant IIV6-458R?

The choice of expression system significantly impacts the quantity, quality, and functionality of recombinant IIV6-458R. Based on current research methodologies, the following expression systems have been evaluated:

Insect cell expression systems (particularly S2 cells from Drosophila) have proven most effective for producing functional IIV6-458R, as they provide the appropriate cellular environment for proper folding and post-translational modifications, including myristoylation . Purification from insect cells typically involves ultracentrifugation for concentration, followed by low-speed centrifugation (1500 × g, 10 min, 4°C) to remove cellular debris .

For studies requiring high yields, an optimized protocol using S2 cells with inducible promoters and subsequent purification by ultracentrifugation (60,000× g, 2 h, 4°C) has demonstrated superior results .

What methods are recommended for studying IIV6-458R interactions with host cell components?

Several complementary techniques are recommended for investigating IIV6-458R interactions with host cellular components:

• Co-immunoprecipitation (Co-IP):

  • Use anti-IIV6-458R antibodies to pull down protein complexes

  • Identify interacting partners by mass spectrometry

  • Validates direct protein-protein interactions

• Confocal Microscopy:

  • Prepare samples by seeding cells onto coverslips

  • Fix with 4% paraformaldehyde (20 minutes)

  • Permeabilize with 0.1% Triton-X

  • Use fluorescently-labeled antibodies against IIV6-458R

  • Co-stain with markers for cellular compartments

  • Analyze co-localization patterns

• Transmission Electron Microscopy (TEM):

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

  • Dehydrate in ethanol, infiltrate with Spurrs' resin

  • Section and stain with 4% uranyl acetate and Reynolds lead

  • Visualize on a transmission electron microscope

• Proximity Ligation Assays:

  • Detect protein interactions with high specificity and sensitivity

  • Particularly useful for membrane-associated proteins

  • Provides spatial resolution of interaction sites

Researchers should combine multiple approaches to validate interactions, as membrane protein associations can be transient and context-dependent .

How can researchers verify the structural integrity of purified recombinant IIV6-458R?

Verifying the structural integrity of purified IIV6-458R is critical for functional studies. Recommended methodologies include:

• Circular Dichroism (CD) Spectroscopy:

  • Assess secondary structure composition

  • Monitor protein folding and stability

  • Compare spectral profiles with predicted structural models

• Size Exclusion Chromatography (SEC):

  • Evaluate protein homogeneity and oligomeric state

  • Detect aggregation or degradation

  • Can be coupled with multi-angle light scattering (SEC-MALS) for precise molecular weight determination

• Limited Proteolysis:

  • Probe the accessibility of protease cleavage sites

  • Correctly folded membrane proteins typically show resistance to digestion in transmembrane regions

  • Compare digestion patterns with in silico predictions

• Fluorescence Spectroscopy:

  • Measure intrinsic tryptophan fluorescence

  • Assess tertiary structure integrity

  • Monitor changes upon ligand binding or environmental conditions

• Western Blot Analysis:

  • Use specific antibodies to verify protein identity

  • Confirm expected molecular weight

  • Detect potential degradation products

A multi-method approach is strongly recommended, as no single technique provides comprehensive structural validation for membrane proteins.

What is known about the functional role of IIV6-458R in viral replication and host interactions?

The IIV6-458R protein is classified among the core IIV genes involved in virion formation, specifically as a myristoylated membrane protein . Current research indicates several key functions:

• Virion Assembly: As a structural component, IIV6-458R contributes to the assembly of viral particles. The protein's myristoylation likely facilitates membrane association during virion formation.

• Membrane Interaction: The protein's structure suggests it participates in the formation of the viral lipid membrane, which is composed predominantly of phosphatidylinositol and diglycerides, differing significantly from host cell membranes .

• Host Range Determination: Comparative genomic analyses between invertebrate and vertebrate iridoviruses suggest that IIV6-458R may contribute to host specificity, as it is present in IIV-6 but not in vertebrate iridoviruses .

• Immune Response Modulation: Studies have shown that IIV-6 can stimulate immune responses in both invertebrate and vertebrate systems. IIV6-458R, as a membrane protein, may be exposed on the virion surface and potentially interact with host immune receptors .

Research using RNA interference (RNAi) and gene knockout approaches has demonstrated that viral membrane proteins, including IIV6-458R, are essential for productive infection cycles, as they mediate critical virus-host interactions during entry and assembly phases .

How does IIV6-458R compare structurally and functionally to similar proteins in other viruses?

Comparative analysis of IIV6-458R with similar proteins in related viruses provides insights into its evolutionary conservation and functional significance:

The absence of clear homologs in vertebrate iridoviruses suggests that IIV6-458R may contribute to the invertebrate host specificity of IIV-6. Phylogenetic analyses indicate that IIV-3 (the sole member of the genus Chloriridovirus) is distantly related to other iridovirus genera, which is reflected in the moderate sequence identity between their membrane proteins .

This divergence suggests that while the basic function of membrane association is conserved, the specific host interactions and structural roles may differ significantly across the family Iridoviridae .

What evidence exists for the role of IIV6-458R in host immune response modulation?

Studies investigating the interaction between IIV-6 and host immune systems have provided several lines of evidence regarding IIV6-458R's potential immunomodulatory roles:

• RNA-based Immune Responses: Although IIV-6 is a DNA virus, research has shown that it activates RNA-sensing pathways in both invertebrate and vertebrate hosts. In Drosophila, IIV-6 is targeted by an RNAi-based antiviral immune response, in which viral replication intermediates are processed by Dicer-2 into viral small interfering RNAs (vsiRNAs) .

• Mammalian Innate Immune Activation: Remarkably, IIV-6 can stimulate a type I interferon-dependent antiviral immune response in mammalian cells. This immune response is mediated by the RIG-I-like receptor (RLR) pathway rather than the canonical DNA sensing pathway via cGAS/STING .

• Cross-Protection Against Arboviruses: The RLR-driven mammalian innate immune response to IIV-6 can protect cells from subsequent infection with arboviruses like Vesicular Stomatitis virus and Kunjin virus, suggesting potential applications in understanding cross-protective immunity .

• Protein-specific Effects: As a membrane protein, IIV6-458R may be exposed on the virion surface and recognized by pattern recognition receptors. Experimental evidence shows phosphorylation of IRF3 and translocation of NFκB in response to IIV-6 infection, processes that may involve recognition of viral membrane components .

These findings suggest that IIV6-458R, as part of the viral structure, may play a role in the initial recognition of the virus by host immune systems, potentially triggering antiviral responses through both RNA and protein-sensing pathways.

How can codon usage analysis of the IIV6-458R gene inform evolutionary and functional studies?

Codon usage bias (CUB) analysis of the IIV6-458R gene provides valuable insights into evolutionary processes and host adaptation strategies:

• Evolutionary Pressure Indicators: Analysis of relative synonymous codon usage (RSCU) values can reveal evolutionary pressures acting on the gene. The topoisomerase II gene in IIV-6 has been studied as a model, showing that when RSCU values exceed 1.6 or fall below 0.6, this indicates codons that are overrepresented or underrepresented, respectively .

• Host Adaptation Mechanisms: The Codon Adaptation Index (CAI) can predict the ability of viral genes to adapt to their host organisms. By comparing the CAI values of IIV6-458R against reference codon usage patterns from host species, researchers can assess potential translational efficiency .

• Dinucleotide Bias Analysis: Examining dinucleotide relative frequencies in the IIV6-458R gene can reveal host-specific signatures. When the value of ρxy falls below 0.78 or exceeds 1.23, it indicates underrepresented or overrepresented dinucleotides, which may reflect adaptation to host CpG suppression patterns .

• Correspondence Analysis (COA): This multivariate statistical technique can examine the main trend of variability within codon usage across genes. COA using RSCU values of 59 sense codons reveals patterns associated with nucleotide content in the 3rd codon position, potentially indicating selection pressure points .

Researchers can apply these methodologies specifically to IIV6-458R to understand how this membrane protein gene has evolved in relation to host adaptation and functional constraints.

What approaches are recommended for studying the role of IIV6-458R in cross-species transmission?

Investigating IIV6-458R's role in cross-species transmission is particularly relevant given that IIV-6 has been detected in both invertebrate and vertebrate hosts. Recommended approaches include:

• Comparative Infectivity Studies:

  • Generate recombinant viruses with modified IIV6-458R genes

  • Test infection efficiency in various host species (both invertebrate and vertebrate)

  • Compare viral titers, replication kinetics, and host responses

  • Example experimental design: Inoculate adult Drosophila flies intraabdominally with IIV-6 variants and monitor survival and viral titers over time

• Host-switch Modeling:

  • Examine natural transmission between predator-prey relationships

  • Analyze viral adaptation following host switching

  • Document genetic changes in the IIV6-458R gene after passage in different hosts

  • Previous research has postulated a host-switch of this virus from prey insects to predator lizards

• Receptor Binding Studies:

  • Identify potential host receptors for IIV6-458R

  • Compare receptor binding affinity across different species

  • Use surface plasmon resonance or biolayer interferometry techniques

• Structural Adaptation Analysis:

  • Compare IIV6-458R sequences isolated from different host species

  • Identify adaptive mutations that correlate with host range expansion

  • Model structural changes that might facilitate new host interactions

• Animal Infection Models:

  • Use established models like bearded dragons (Pogona vitticeps) for vertebrate studies

  • Compare with insect models like crickets

  • Assess transmission dynamics and pathogenicity

  • Previous studies have shown that IIV isolates from lizard hosts are capable of infecting crickets, demonstrating cross-species transmission potential

These approaches can provide mechanistic insights into how IIV6-458R may contribute to the virus's ability to cross species barriers, a phenomenon observed between invertebrate hosts and poikilothermic vertebrates.

What potential applications exist for using IIV6-458R in biotechnology and therapeutic development?

IIV6-458R has several potential applications in biotechnology and therapeutic development:

• Adjuvant Development:

  • IIV-6 has been shown to stimulate innate immune responses in mammalian cells

  • IIV6-458R, as a membrane protein, may contribute to this immunostimulatory effect

  • Purified or modified versions could be developed as adjuvants for vaccines

  • Particularly promising for enhancing responses against arboviruses, as IIV-6 has demonstrated protective effects against subsequent arboviral infections

• Membrane Protein Expression Platform:

  • The successful expression and purification systems developed for IIV6-458R could serve as a platform for other challenging membrane proteins

  • The protein's ability to incorporate into lipid membranes could be exploited for liposome or nanoparticle formulations

• Antiviral Target Identification:

  • Understanding IIV6-458R's role in virus assembly and host interaction could reveal common mechanisms exploited by related viruses

  • These insights may identify novel targets for broad-spectrum antiviral development

• Diagnostic Applications:

  • Recombinant IIV6-458R could be used in the development of serological assays for detecting anti-iridovirus antibodies

  • Particularly useful for environmental monitoring and wildlife disease surveillance

• CRISPR-based Gene Editing Tools:

  • Viral membrane fusion mechanisms could inspire the development of new cell-penetrating delivery systems for gene editing components

  • IIV6-458R's membrane-interacting properties might be adapted for enhanced cellular delivery

• Cross-protective Immunity Studies:

  • The observation that IIV-6 can protect cells from subsequent arbovirus infection suggests potential for developing cross-protective immunization strategies

  • IIV6-458R could be a key component in understanding and exploiting this cross-protection mechanism

These applications leverage the unique properties of IIV6-458R and the increasing understanding of its structural and functional characteristics in both invertebrate and vertebrate systems.

What are the common challenges in producing high-quality recombinant IIV6-458R and how can they be addressed?

Researchers face several technical challenges when working with recombinant IIV6-458R, primarily due to its nature as a membrane protein:

For virus propagation specifically, researchers have successfully used 175 cm² tissue culture flasks with appropriate cell lines, followed by purification and concentration via ultracentrifuge pelleting in a Beckman XL-90 instrument using a 60 Ti rotor (60,000× g, 2 h, 4°C) .

How can researchers address data inconsistencies when studying IIV6-458R function across different experimental systems?

When studying IIV6-458R across different experimental systems, researchers may encounter inconsistent results due to various factors. Addressing these inconsistencies requires systematic troubleshooting:

• Standardize Protein Preparation:

  • Implement consistent expression and purification protocols

  • Validate protein folding and post-translational modifications

  • Use the same buffer conditions across experiments

  • Consider creating a reference standard batch for calibration

• Control Host Cell Variables:

  • Document passage number of cell lines

  • Standardize culture conditions (media, supplements, confluence)

  • Account for host cell type differences in data interpretation

  • Include appropriate cellular controls for each experimental system

• Normalize Infection Parameters:

  • Standardize multiplicity of infection (MOI) calculations

  • Use the same viral stock for comparative experiments

  • Consider implementing qPCR-based quantification of viral loads

  • Previous studies have used MOI of 1 TCID₅₀/cell for consistent results

• Address Technical Variability:

  • Implement biological and technical replicates

  • Use statistical methods appropriate for non-normal distributions

  • Develop internal standards for each assay type

  • Consider blinding during data collection and analysis

• Document Environmental Conditions:

  • Record temperature fluctuations during experiments

  • Standardize incubation conditions (28°C has been used for IIV-6 culture)

  • Account for media pH and oxygen tension

  • Control for light exposure in fluorescence-based assays

By systematically controlling these variables and thoroughly documenting experimental conditions, researchers can better identify the source of inconsistencies and develop more robust experimental designs.

What is the current state of research on IIV6-458R and what are the critical knowledge gaps?

The current state of research on IIV6-458R reveals several critical knowledge gaps that represent opportunities for future investigation:

Current Knowledge:

• IIV6-458R is classified as a putative myristoylated membrane protein involved in virion formation

• The protein's amino acid sequence and basic structural features have been characterized

• IIV6-458R is present in invertebrate iridoviruses but absent in vertebrate iridoviruses

• The protein likely contributes to the formation of the viral lipid membrane, which has a distinctive composition compared to host cell membranes

Critical Knowledge Gaps:

Addressing these knowledge gaps would significantly advance our understanding of IIV6-458R's role in viral biology and potentially lead to novel applications in biotechnology and therapeutics.