Recombinant Invertebrate iridescent virus 6 Putative membrane protein 337L (IIV6-337L)

What is Invertebrate iridescent virus 6 (IIV6) and how is membrane protein 337L situated within its structure?

IIV6 is a large dsDNA virus belonging to the family Iridoviridae, specifically classified within the genus Iridovirus in the Betairidovirinae subfamily. It primarily infects invertebrate ectotherms and causes symptoms ranging from minor fitness reductions to systemic disease with high mortality . The virus derives its name from the iridescence observed in infected hosts, which results from light reflection by paracrystalline arrays of virus particles .

The virion structure of IIV6 comprises three concentric domains:

• An outer proteinaceous capsid layer with icosahedral symmetry (T = 147 lattice)

• An intermediate lipid membrane containing associated proteins

• A central nucleocytoplasmic dsDNA genome

IIV6-337L is a myristylated membrane protein associated with the intermediate lipid membrane. This membrane has a unique composition predominantly featuring phosphatidylinositol and diglycerides, which differs significantly from host cell membranes. The major constituent, glycophosphatidylinositol (GPI), acts as a lipid anchor for membrane proteins . As a myristylated protein, IIV6-337L likely has a fatty acid modification that helps anchor it within this specialized viral membrane.

How does IIV6-337L relate to other membrane proteins in the Iridoviridae family?

Phylogenetic analyses indicate that IIV6-337L (NP_149800.1) has orthologous proteins in other iridoviruses, specifically DIV1_074R and DIV1_302R in Daphnia iridescent virus 1 (DIV-1). The degree of conservation varies depending on analysis parameters, with algebraic connectivity ranging from 9.2% to 41.5% . This moderate level of conservation suggests that while the protein serves important functions across iridoviruses, it may have evolved species-specific adaptations.

The table below shows the comparison of IIV6-337L with its orthologs in DIV-1 across different clustering analyses:

For context, highly conserved proteins like the major capsid protein (MCP) show 100% algebraic connectivity across all analyses, while IIV6-337L demonstrates more variation, suggesting it may have undergone more extensive evolutionary adaptation .

What transcriptional regulation governs IIV6-337L expression during viral infection?

IIV6 genes are temporally regulated and divided into three kinetic classes: immediate-early (IE), delayed-early (DE), and late (L) genes. Each class is controlled by specific promoter motifs in their upstream regions . While the specific transcriptional class of IIV6-337L is not explicitly mentioned in the available literature, understanding its classification would provide insights into its role in the viral life cycle.

For immediate-early genes, the conserved promoter motif AA(A/T)(T/A)TG(A/G)A has been identified, while delayed-early genes feature the (T/A/C)(T/G/C)T(T/A)ATGG motif. Late genes contain the motifs (T/G)(C/T)(A/C)A(T/G/C)(T/C)T(T/C) and (C/G/T)(G/A/C)(T/A)(T/G)(G/T)(T/C), though interestingly, the latter appears to function as a repressor sequence .

Based on the general patterns observed with viral membrane proteins, IIV6-337L likely belongs to the late gene class, as structural proteins involved in virion assembly are typically expressed during the late phase of infection.

What structural features can be predicted for IIV6-337L based on computational analysis?

As a myristylated membrane protein, IIV6-337L likely contains several key structural elements:

• A myristoylation site at the N-terminus (typically at a glycine residue at position 2)

• One or more hydrophobic transmembrane domains that span the viral lipid membrane

• Cytoplasmic and/or luminal domains that may interact with other viral or host proteins

• Potential protein-protein interaction motifs

Computational prediction using tools such as TMHMM, Phobius, or TOPCONS would likely identify the transmembrane regions, while myristoylation prediction tools like NMT or GPS-Lipid could confirm the N-terminal modification site. For tertiary structure prediction, approaches such as AlphaFold, I-TASSER, or SWISS-MODEL could generate preliminary models, though these would require experimental validation.

The lipid environment of IIV6-337L is particularly noteworthy, as the viral membrane contains a high proportion of glycophosphatidylinositol (GPI), which serves as a lipid anchor for membrane proteins. This composition differs significantly from host cell membranes, suggesting that IIV6-337L may have evolved specific adaptations for this unique lipid environment .

How can the structure-function relationship of IIV6-337L be experimentally determined?

Determining the structure-function relationship of IIV6-337L requires a multi-faceted approach combining structural biology techniques with functional assays:

Structural studies:

• NMR spectroscopy: Solution NMR can be used for smaller domains, while solid-state NMR is suitable for membrane-embedded regions. From the study of Vpu (another viral membrane protein), we see that combining solution NMR on phospholipid micelles with solid-state NMR on oriented phospholipid bilayers can yield valuable insights .

• Cryo-electron microscopy (cryo-EM): For full-length protein or protein complexes, particularly within the context of the entire virion.

• X-ray crystallography: Though challenging for membrane proteins, techniques like lipid cubic phase crystallization have proven successful for numerous membrane proteins.

Functional studies:

• Mutagenesis: Systematic mutation of key residues to identify those critical for function. This could include:

  • Alanine scanning

  • Deletion of predicted functional domains

  • Mutation of the myristoylation site

• Viral replication assays: Comparing wild-type virus with mutants to assess the impact on viral fitness.

• Protein-protein interaction assays: Identifying viral or host proteins that interact with IIV6-337L.

• Membrane association studies: Determining how mutations affect incorporation into viral membranes.

A study on Vpu demonstrates how structural information can be correlated with function: "NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic helices... In agreement with the structural model, truncated Vpu 2–51, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu 28–81, which lacks the transmembrane helix, does not" .

What is known about the potential interactions between IIV6-337L and host immune pathways?

While direct evidence for IIV6-337L interactions with host immune pathways is limited, IIV6 as a virus has been shown to inhibit NF-κB signaling pathways in Drosophila. Specifically, IIV6 suppresses antimicrobial peptide (AMP) gene induction downstream of both the Imd and Toll pathways .

The mechanism of this inhibition appears to occur downstream of key signal transduction events: "Cleavage of both Imd and Relish, as well as Relish nuclear translocation, three key points in Imd signal transduction, occur in IIV-6 infected cells, indicating that the mechanism of viral inhibition is farther downstream, at the level of Relish promoter binding or transcriptional activation" .

As a membrane protein, IIV6-337L could potentially:

• Interact with host membrane proteins involved in immune signaling

• Disrupt membrane-associated signaling complexes

• Alter membrane properties in ways that affect receptor clustering or signal transduction

• Participate in the formation of viral replication complexes that sequester host factors

Investigating whether IIV6-337L contributes to the observed immune evasion would require experiments such as:

• Expression of IIV6-337L alone in cells and assessment of NF-κB pathway activity

• Comparison of wild-type IIV6 with IIV6-337L mutants for their ability to inhibit immune responses

• Identification of host proteins that interact with IIV6-337L using techniques like co-immunoprecipitation or proximity labeling

What expression systems are optimal for producing recombinant IIV6-337L for structural and functional studies?

The choice of expression system for IIV6-337L should consider the unique challenges of membrane protein expression. Several options, with their respective advantages and limitations, are outlined below:

1. Insect cell expression systems:

• Advantages: Most native-like environment for IIV6 proteins, proper post-translational modifications including myristoylation, natural lipid composition closer to viral environment

• Specific systems: Sf9, Sf21, or High Five cells with baculovirus expression vector systems (BEVS)

• Considerations: Codon optimization for insect cells, inclusion of purification tags (His, FLAG, etc.)

2. Yeast expression systems:

• Advantages: Eukaryotic folding machinery, capability for post-translational modifications, lower cost than insect or mammalian systems

• Specific systems: Pichia pastoris, Saccharomyces cerevisiae

• Considerations: Different membrane composition than insect cells, potential hyperglycosylation

3. Cell-free expression systems:

• Advantages: Direct incorporation into provided lipid environments, avoids toxicity issues, rapid production

• Specific systems: Insect cell extracts with supplied lipids or nanodiscs

• Considerations: Lower yield, higher cost, limited post-translational modifications

4. Bacterial systems (less optimal):

• Advantages: High yield, low cost, well-established protocols

• Specific systems: E. coli strains specialized for membrane proteins (C41/C43)

• Limitations: Lack of eukaryotic post-translational modifications, different membrane composition, protein often forms inclusion bodies

For IIV6-337L, the baculovirus expression system in insect cells represents the most physiologically relevant approach, as it provides:

• The capability for myristoylation

• A lipid environment more similar to the virus's natural context

• Appropriate folding machinery for a protein from an insect virus

To facilitate purification and structural studies, the recombinant protein should include:

• Affinity tags positioned to minimize interference with function

• Protease cleavage sites for tag removal

• Potentially, fusion partners to enhance solubility or crystallization

What are the most effective methods for studying protein-protein interactions involving IIV6-337L?

Understanding the interactome of IIV6-337L is crucial for elucidating its function. Several complementary approaches can be employed:

1. Proximity-based labeling techniques:

• BioID: Fusion of IIV6-337L with a biotin ligase (BirA*) to biotinylate proximal proteins

• APEX2: Fusion with an engineered peroxidase that catalyzes biotin-phenol labeling of nearby proteins

• Advantages: Captures transient interactions in native cellular environment, works well for membrane proteins

• Workflow: Express fusion protein in cells → Activate labeling → Lyse cells → Purify biotinylated proteins → Identify by mass spectrometry

2. Co-immunoprecipitation (Co-IP) and pull-down assays:

• Traditional Co-IP: Using antibodies against IIV6-337L to precipitate interaction partners

• Tagged pull-down: Using recombinant tagged IIV6-337L to pull down interaction partners

• Crosslinking Co-IP: Addition of chemical crosslinkers to stabilize transient interactions

• Advantages: Relatively straightforward, can validate individual interactions

3. Yeast two-hybrid variants for membrane proteins:

• Split-ubiquitin yeast two-hybrid: Specifically designed for membrane protein interactions

• Membrane yeast two-hybrid (MYTH): Detects interactions with membrane-bound proteins

• Advantages: High-throughput screening capability, specific for membrane proteins

4. Quantitative interaction proteomics:

• SILAC or TMT labeling: For quantitative comparison of interaction partners between conditions

• Advantages: Provides quantitative data on interaction strength, can distinguish specific from non-specific interactions

5. Biophysical methods for direct interaction measurement:

• Surface plasmon resonance (SPR): Measures real-time binding kinetics between purified components

• Microscale thermophoresis (MST): Detects interactions through changes in thermophoretic mobility

• Advantages: Provides quantitative binding parameters (Kd, kon, koff), requires purified components

Experimental design considerations:

• Include appropriate controls (GFP fusions, non-myristoylated mutants, etc.)

• Validate key interactions through multiple independent methods

• Consider membrane environment effects on interactions

• Test interactions in the context of viral infection when possible

How can advanced genomic technologies be applied to study IIV6-337L function?

Contemporary genomic technologies offer powerful approaches to investigate IIV6-337L function in a comprehensive manner:

1. CRISPR-Cas9 genome editing for viral manipulation:

• Gene knockout: Generate IIV6 variants lacking the 337L gene to assess essentiality

• Domain mutations: Introduce specific mutations to test the function of predicted domains

• Reporter integration: Insert fluorescent tags to monitor protein localization and dynamics

• Workflow: Design guide RNAs targeting IIV6-337L → Clone into CRISPR vectors → Transfect along with viral genome → Screen for edited viruses → Characterize phenotypes

2. CRISPR screening for host factor identification:

• Genome-wide screens: Identify host factors required for IIV6-337L function

• Focused screens: Target specific pathways (e.g., NF-κB signaling) to identify relevant interactions

• Approaches:

  • Loss-of-function screens with CRISPR knockout libraries

  • Gain-of-function screens with CRISPR activation

• Readouts: Viral replication efficiency, reporter assays for specific pathways

3. Next-generation sequencing applications:

• RNA-Seq: Compare transcriptome changes between wild-type and IIV6-337L mutant viruses

• CLIP-Seq: If IIV6-337L interacts with RNA, identify binding sites

• Ribosome profiling: Examine impacts on host translation

4. Systems biology approaches:

• Interactome mapping: Combine protein-protein interaction data with functional genomics

• Network analysis: Identify key host pathways affected by IIV6-337L

• Multi-omics integration: Combine proteomics, transcriptomics, and metabolomics data

5. Optimal experimental design through computational modeling:
The minimally sufficient experimental design approach described by Gevertz and Kareva could be applied to IIV6-337L research:

• Develop mathematical models of IIV6-337L function

• Use identifiability analysis to determine the minimal set of measurements needed

• Optimize experimental design to ensure parameter identifiability while minimizing experimental costs

This systematic approach would allow researchers to "identify a minimal set of time points when data needs to be collected that robustly ensures practical identifiability of model parameters" , making IIV6-337L research more efficient and conclusive.

How does research on IIV6-337L contribute to understanding broader principles of viral membrane protein function?

Research on IIV6-337L offers several valuable contributions to the field of viral membrane proteins:

1. Evolutionary insights:

• IIV6-337L shows moderate conservation with orthologs in other iridoviruses (e.g., DIV-1), with algebraic connectivity ranging from 9.2% to 41.5% . This pattern of conservation helps identify functionally critical domains versus adaptable regions.

• Comparing IIV6-337L with membrane proteins from other viral families could reveal convergent evolutionary strategies for membrane interaction and host manipulation.

2. Structure-function relationships in specialized viral membranes:

• The IIV6 lipid membrane has a unique composition, predominantly featuring phosphatidylinositol and diglycerides, which differs from host cell membranes .

• Understanding how IIV6-337L functions within this specialized membrane environment provides insights into membrane protein adaptation to non-standard lipid compositions.

3. Host-pathogen interface principles:

• As IIV6 inhibits NF-κB signaling pathways in host cells , investigating whether IIV6-337L contributes to this immune evasion could reveal conserved mechanisms by which viral membrane proteins disrupt host signaling.

• The relationship between membrane localization and immune evasion represents a fundamental aspect of virus-host interaction.

4. Model for studying membrane protein biogenesis:

• Studies on IIV6-337L could illuminate how viral membrane proteins are synthesized, folded, and integrated into viral membranes.

• Drawing parallels to cellular membrane protein folding mechanisms, such as those studied in YidC-dependent membrane protein insertion , could identify conserved or divergent principles.

5. Contribution to classification and taxonomy:

• Membrane proteins like IIV6-337L provide phylogenetic information that helps resolve ambiguous relations between viral isolates .

• Molecular characterization of IIV6-337L contributes to the ongoing refinement of iridovirid classification, which has been challenging due to the continuous discovery of novel strains.

What implications does IIV6-337L research have for understanding host range and viral tropism?

IIV6-337L research offers valuable insights into the determinants of host range and tissue tropism:

1. Role in host specificity:

• Membrane proteins often mediate virus-host interactions that determine host range.

• While invertebrate iridoviruses (IIVs) primarily infect invertebrates, they "can occasionally infect vertebrates; thus, host range is often not a useful criterion for classification" . The molecular basis for this cross-species potential could involve membrane proteins like IIV6-337L.

2. Experimental evidence from infection models:

• Infection trials with IIV-6 have demonstrated significant variation in susceptibility among host genotypes. In one study, infection rates ranged from 0% to 78% among different host genotypes, with Finnish clones from the same site as the virus showing particularly high infection rates (46% and 78%) .

• This variation suggests genetic determinants of susceptibility that might involve interactions with viral membrane proteins.

3. Comparative analysis with other iridoviruses:

• The genomic differences between IIV6-337L and its orthologs in other iridoviruses could help explain differences in host range.

• For instance, the Daphnia iridescent virus 1 (DIV-1) encodes a suite of unique proteins not found in other iridoviruses, potentially contributing to its specific host tropism .

4. Infectious process insights:

• In Drosophila, IIV-6 infection results in visible iridescence in the eyes, thorax, and abdomen, "which is the result of light reflection by assemblies of paracrystalline arrays of IIV-6 particles" .

• Understanding how IIV6-337L contributes to virion assembly and the formation of these paracrystalline arrays could explain tissue-specific manifestations.

5. Co-infection dynamics:

• Flies co-infected with both IIV-6 and the Gram-negative bacterium Erwinia carotovora carotovora "succumb to infection more rapidly than flies singly infected with either the virus or the bacterium" .

• This observation highlights the importance of studying how viral membrane proteins like IIV6-337L might interact with bacterial co-infections, potentially through altered immune responses.

How can advanced structural biology methods be optimized for studying the membrane integration and topology of IIV6-337L?

Studying membrane protein topology and integration requires specialized approaches that address the challenges of membrane environments:

1. Cryo-electron microscopy (cryo-EM) strategies:

• Sample preparation optimization:

  • Reconstitution in nanodiscs or amphipols to maintain native structure

  • Vitrification conditions that preserve the lipid environment

  • Grid types that minimize protein adsorption to the air-water interface

• Data collection considerations:

  • Use of phase plates to enhance contrast for smaller membrane proteins

  • Movie-mode acquisition to correct for beam-induced motion

  • Tilt series for difficult orientations

• Processing approaches:

  • Symmetry-based reconstruction of IIV6-337L oligomers if present

  • Classification strategies to handle conformational heterogeneity

  • Local resolution refinement for transmembrane regions

2. NMR spectroscopy approaches:

• Solution NMR for soluble domains:

  • Isotopic labeling strategies (15N, 13C, 2H)

  • Domain-focused studies of non-membrane regions

• Solid-state NMR for membrane-embedded regions:

  • Oriented sample solid-state NMR in lipid bilayers

  • Magic-angle spinning methods for structural details

• Combined approach: Following the strategy used for HIV-1 Vpu protein where "NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic helices"

3. Membrane topology mapping:

• Cysteine accessibility methods:

  • Substitution of residues with cysteines and testing accessibility to membrane-impermeable reagents

• Fluorescence approaches:

  • Environment-sensitive fluorophores to detect membrane-embedded versus exposed regions

• Protease protection assays:

  • Determine regions protected by the membrane from proteolytic digestion

4. Experimental design optimization:

• Following the minimally sufficient experimental design framework:

  • "Identify an optimal experimental design (how much data to collect and when to collect it) that ensures parameter identifiability (permitting confidence in model predictions), while minimizing experimental time and costs"

  • Use profile likelihood approaches to determine which measurements provide maximum information

  • Focus resources on the experiments most likely to resolve structural ambiguities

5. Integrative structural biology:

• Combine multiple techniques:

  • Low-resolution envelope from small-angle X-ray scattering (SAXS)

  • Distance constraints from electron paramagnetic resonance (EPR)

  • High-resolution structures of individual domains from X-ray crystallography or NMR

  • Computational modeling to integrate diverse experimental constraints

This multi-technique approach ensures robust structural characterization while acknowledging the inherent challenges of membrane protein analysis.

What are the most promising avenues for future research on IIV6-337L?

Based on current understanding and technological capabilities, several high-priority research directions emerge:

1. Comprehensive functional characterization:

• Generate and characterize IIV6 variants with mutations or deletions in the 337L gene

• Assess the impact on viral replication, assembly, and host range

• Determine whether IIV6-337L contributes to the observed inhibition of NF-κB responses

2. Structural elucidation:

• Determine the high-resolution structure of IIV6-337L using cryo-EM or hybrid methods

• Map membrane topology and identify functional domains

• Characterize potential oligomerization or complex formation

3. Host-interaction network:

• Identify host proteins that interact with IIV6-337L using proximity labeling approaches

• Validate key interactions and determine their functional significance

• Map the interactome in different host species to understand host range determinants

4. Evolution and adaptation studies:

• Compare IIV6-337L with orthologs from other iridoviruses to identify conserved and variable regions

• Investigate how IIV6-337L has adapted to different hosts through experimental evolution studies

• Analyze selection pressures acting on different domains of the protein

5. Development of IIV6-337L as a biotechnological tool:

• Explore potential applications as a membrane protein expression tag or fusion partner

• Investigate use as a model system for studying membrane protein folding and integration

• Assess potential as a target for broad-spectrum antivirals against related viruses of economic importance

How might comparative studies between IIV6-337L and related proteins advance understanding of iridovirus diversity?

Comparative analysis of IIV6-337L with related proteins can provide critical insights into iridovirus evolution and host adaptation:

1. Phylogenetic mapping:

• Using the framework established for IIV classification, where "three major groups in genus Iridovirus were identified: custaceoiridovirus (group 1), oligoiridovirus (group 2), and polyiridovirus (group 3)" , determine how 337L protein sequences correlate with these groupings.

• Assess whether 337L phylogeny aligns with whole-genome phylogeny or shows evidence of horizontal gene transfer or recombination.

2. Structure-function comparative analysis:

• Compare predicted structural features of 337L orthologs across different iridovirus species

• Identify conserved structural elements versus variable regions

• Correlate structural differences with host range and tissue tropism

3. Experimental validation across species:

• Test functional complementation between 337L proteins from different iridoviruses

• Assess whether 337L from one species can function in another

• Identify species-specific interaction partners

4. Molecular evolution analysis:

• Calculate selection pressures (dN/dS ratios) across different domains of 337L

• Identify sites under positive selection that may indicate host adaptation

• Correlate evolutionary rates with host switching events

5. Taxonomic implications:

• Address the need for revision of IIV classification, as "in light of these recent findings, the current IIV classification needs to be revised"

• Contribute molecular data on 337L to help resolve "ambiguous relations" between viral isolates

• Assess whether 337L characteristics align with proposed new taxonomic groupings

This comparative approach would not only advance understanding of iridovirus diversity but also contribute to the broader field of virus evolution and host adaptation.

What technological innovations would most benefit future studies of IIV6-337L?

Several emerging technologies and methodological innovations could significantly advance IIV6-337L research:

1. Advanced imaging techniques:

• Cryo-electron tomography (cryo-ET): For visualizing IIV6-337L in its native context within virions

• Super-resolution microscopy: For tracking IIV6-337L dynamics during infection

• Correlative light and electron microscopy (CLEM): To link functional observations with structural context

2. Single-molecule approaches:

• Single-molecule FRET: To study conformational changes in IIV6-337L

• High-speed AFM: For real-time visualization of membrane protein dynamics

• Optical tweezers: To measure forces involved in membrane integration or protein-protein interactions

3. Advanced protein engineering methods:

• Non-canonical amino acid incorporation: For site-specific labeling of IIV6-337L

• Minimal protein design: To identify the core functional elements of IIV6-337L

• Split protein complementation: For detecting protein-protein interactions in live cells

4. Artificial intelligence and computational methods:

• AI-driven structure prediction: Building on advances like AlphaFold to predict membrane protein structures with higher accuracy

• Molecular dynamics simulations: To model IIV6-337L behavior in membrane environments

• Network analysis algorithms: To interpret complex host-pathogen interaction data

5. Organ-on-chip and advanced cell culture systems:

• Insect tissue-on-chip platforms: For studying IIV6-337L in more physiologically relevant contexts

• 3D organoid cultures: To investigate tissue tropism determinants

• Microfluidic systems: For real-time monitoring of infection dynamics

6. High-throughput functional genomics:

• CRISPR base editing and prime editing: For precise modification of IIV6-337L without double-strand breaks

• Perturb-seq approaches: Combining CRISPR perturbations with single-cell RNA-seq to assess global effects

• Spatial transcriptomics: To map host responses to IIV6-337L with spatial resolution

These technological innovations would enable more precise, comprehensive, and physiologically relevant studies of IIV6-337L, potentially leading to breakthroughs in understanding its structure, function, and role in viral pathogenesis.