Recombinant Invertebrate iridescent virus 6 Uncharacterized protein 140L (IIV6-140L)

What is Invertebrate iridescent virus 6 Uncharacterized protein 140L?

Invertebrate iridescent virus 6 Uncharacterized protein 140L (IIV6-140L) is a viral protein encoded by the IIV6 genome, which belongs to the family Iridoviridae, genus Iridovirus. The protein consists of 64 amino acids with the sequence MNINDYKDSIIVGVLFLLFTRDWFDELIFGTFPSLKGMPWVFLALKVLGIMVLFYLLDAIINVK . While classified as "uncharacterized," the protein is believed to play a role in viral structure or function based on its conservation within the IIV6 genome. IIV6, also known as Chilo iridescent virus, has a large DNA genome approximately 212 kbp in length with 28.6% G+C content and comprises 468 open reading frames (ORFs) . The 140L designation corresponds to its position and orientation within the viral genome annotation system.

How does IIV6-140L compare structurally to other proteins in the IIV6 genome?

The IIV6-140L protein is one of many proteins encoded within the IIV6 genome, which contains both core and non-core genes. Core genes in IIV6 include those encoding major capsid proteins (MCP), ATPases, and DNA polymerases, which are conserved across the Iridoviridae family . In contrast, IIV6-140L falls into the category of uncharacterized proteins with unknown function, distinct from the well-characterized core proteins such as the major capsid protein (MCP) that has been extensively used for phylogenetic analysis. Structural prediction analyses suggest IIV6-140L may contain transmembrane domains, as indicated by its hydrophobic amino acid composition in portions of the sequence. When compared to other IIV6 proteins like the myristilated membrane protein (118L, 458R) or proteins involved in apoptosis inhibition (157L, 193R), IIV6-140L represents a less characterized component of the viral proteome . The protein's relatively small size (64 amino acids) suggests it may serve as an accessory protein rather than a major structural component.

What are the available forms of recombinant IIV6-140L for research applications?

Recombinant IIV6-140L is available as a purified protein preparation primarily for ELISA and other immunological applications. Commercial preparations typically provide the protein at a concentration suitable for experimental use (approximately 50 μg per vial) in a Tris-based buffer with 50% glycerol, optimized for protein stability . The recombinant protein is generally produced with an affinity tag, though the specific tag type may vary depending on the production process and intended application. For long-term storage, recombinant IIV6-140L should be kept at -20°C or -80°C, with working aliquots maintained at 4°C for up to one week to avoid degradation from repeated freeze-thaw cycles . Researchers can also request custom preparations with different quantities or buffer formulations depending on specific experimental needs.

What are the best experimental approaches to study IIV6-140L function?

The optimal experimental approach to study IIV6-140L function involves a multi-faceted strategy combining both in vitro and in vivo methodologies. Initially, researchers should establish expression systems for producing recombinant IIV6-140L with various tags (His, GST, or FLAG) to facilitate protein purification and interaction studies . Protein-protein interaction assays, including co-immunoprecipitation and yeast two-hybrid screening, can identify binding partners within both viral and host proteomes, providing insights into potential functional networks. For in vivo studies, generating recombinant IIV6 viruses with mutations or deletions in the 140L gene using CRISPR-Cas9 or homologous recombination techniques would allow for comparative phenotypic analyses. Cell culture infection models using both invertebrate cell lines (such as Drosophila S2 cells) and vertebrate cell lines can help determine if the protein plays a role in host range determination or immune evasion . Monitoring viral replication kinetics, transcriptomics, and proteomics in the presence or absence of functional 140L protein provides comprehensive insights into its biological significance.

What cell culture systems are appropriate for studying IIV6-140L interactions?

For studying IIV6-140L interactions, both invertebrate and vertebrate cell culture systems provide valuable but distinct experimental platforms. Invertebrate cell lines, particularly Drosophila S2 cells, represent the natural host environment and should be maintained at 28°C to optimize viral replication and protein expression . Viper heart cells (VH-2) have been successfully used to isolate and propagate IIV6 from reptilian sources and can be employed to study potential vertebrate-invertebrate host transitions . For examining mammalian immune responses to IIV6-140L, human cell lines such as A549 (lung epithelial) and HEK 293T cells are appropriate, as these have demonstrated capacity to mount interferon responses against IIV6 infection . When designing co-culture experiments, it's important to optimize media conditions and temperature settings (typically 28°C for invertebrate cells and 37°C for mammalian cells), which may require compromise temperatures for co-culture systems. For quantifying viral replication in these systems, researchers should employ qPCR targeting viral genomic DNA to track replication kinetics across multiple time points post-infection .

How should experiments be designed to assess potential immune responses to IIV6-140L?

Experiments to assess immune responses to IIV6-140L should incorporate both in vitro cellular assays and in vivo model systems with appropriate controls. For in vitro assessment, researchers should expose various immune cell types (including macrophages and dendritic cells) to purified recombinant IIV6-140L and measure cytokine production (particularly type I interferons) using ELISA assays . Reporter cell lines containing interferon-stimulated response elements (ISRE) coupled to luciferase can quantify interferon signaling activation in response to IIV6-140L exposure. For mechanistic studies, knockout cell lines deficient in specific pattern recognition receptors (PRRs) like RIG-I should be employed to determine which immune pathways recognize the protein or its associated nucleic acids . In vivo, mouse models can be used to assess systemic immune responses following administration of recombinant IIV6-140L, with careful monitoring of interferon production, immune cell activation, and potential inflammatory responses. When designing these experiments, researchers must include appropriate positive controls (such as poly(dA:dT) for DNA sensing pathways) and negative controls (such as unrelated viral proteins) to establish specificity of the immune response to IIV6-140L .

What is currently known about the role of IIV6-140L in viral replication?

The specific role of IIV6-140L in viral replication remains largely uncharacterized, representing a significant gap in our understanding of IIV6 biology. Based on sequence analysis, the protein's hydrophobic regions suggest it may function as a membrane-associated protein potentially involved in virion assembly, budding, or host membrane manipulation . Unlike well-characterized IIV6 proteins such as the major capsid protein (MCP) or DNA polymerase, the 140L protein has not been definitively linked to specific stages of the viral replication cycle. To investigate its role, researchers have employed genomic approaches comparing the complete genome sequence of IIV6 (approximately 212 kbp) against related iridoviruses, identifying potential recombination sites and gene insertions that may affect viral fitness . Targeted gene knockout or silencing experiments would provide the most direct evidence of 140L's function, though such studies are currently limited in the published literature. Future research should focus on temporal expression patterns of 140L during infection and co-localization with known viral replication components to better define its functional significance.

How does IIV6-140L potentially interact with host immune responses?

While the specific immune interactions of IIV6-140L have not been fully characterized, studies of IIV6 provide important context for understanding potential mechanisms. IIV6 has been shown to stimulate a type I interferon-dependent antiviral immune response in mammalian cells despite being an invertebrate DNA virus . Intriguingly, this immune activation occurs not through the canonical cGAS/STING DNA sensing pathway but via the RIG-I-like receptor (RLR) pathway typically associated with RNA virus detection . The role of IIV6-140L within this immune stimulation context requires investigation, as it may either contribute to immune activation or function as an immune evasion factor. Experimental approaches should include comparing immune responses between wild-type IIV6 and 140L-deficient mutants, assessing whether recombinant 140L protein alone can stimulate immune signaling, and identifying potential host protein interactions using immunoprecipitation followed by mass spectrometry. Since RNA polymerase III-mediated transcription of viral DNA has been implicated in RLR activation during IIV6 infection, researchers should investigate whether the 140L gene region is transcribed by this pathway and contributes to immune detection .

How does IIV6-140L compare to homologous proteins in other iridoviruses?

Comparative analysis of IIV6-140L with homologous proteins in other iridoviruses reveals important evolutionary relationships and functional implications. Phylogenetic analysis based on complete genome sequencing shows that IIV6-140L belongs to a group of proteins with varying degrees of conservation across the Iridoviridae family . When comparing IIV6 to lizard-cricket IV isolates (Liz-CrIV), phylogenetic trees based on the major capsid protein (MCP) gene show a considerably longer evolutionary distance than expected, suggesting differing evolutionary pressures across the viral genome including potential regions containing the 140L gene . Sequence alignment of homologous proteins to IIV6-140L across various isolates reveals conserved and variable regions, with the hydrophobic domains typically showing higher conservation, suggesting functional constraints on these structural elements. Studies have identified several deletions, recombination sites, and insertions of genes of non-iridoviral origin when comparing IIV6 with related viruses, highlighting the dynamic evolutionary history of these viral genomes . This comparative approach can help identify which regions of 140L are essential for function versus those that may be adaptable to different host environments.

What evolutionary insights can be gained from studying IIV6-140L?

The evolutionary study of IIV6-140L provides valuable insights into viral adaptation across diverse host ranges and environmental conditions. Analysis of selection pressures acting on the 140L gene sequence can reveal whether it is undergoing purifying selection (conservation) or positive selection (adaptation), indicating its functional importance to viral fitness . Comparative genomics across iridovirus isolates from different hosts (including reptiles, amphibians, and various invertebrates) demonstrates the remarkable host range flexibility of these viruses, with potential implications for the role of proteins like 140L in host switching events . Recombination analysis and identification of horizontal gene transfer events involving the genomic region containing 140L could illuminate how iridoviruses acquire novel functions and adapt to new ecological niches. The study of IIV6-140L in the context of host-virus co-evolution is particularly relevant given the documented cases of IIV transitions between invertebrate and vertebrate hosts, suggesting proteins like 140L may play roles in overcoming species barriers . Understanding these evolutionary dynamics has broader implications for predicting viral emergence and host range expansion in related virus families.

How can IIV6-140L be utilized as a tool in immunological research?

The unique properties of IIV6-140L present several opportunities for its application as a tool in immunological research. As IIV6 has been shown to activate the RIG-I pathway despite being a DNA virus, recombinant IIV6-140L can be employed to investigate cross-talk between different pattern recognition receptor systems in innate immunity . Researchers can develop IIV6-140L-based probes to study the specificity and sensitivity of immune detection mechanisms across different species, providing evolutionary insights into conserved and divergent aspects of antiviral immunity. The protein can be incorporated into experimental vaccine platforms as either an adjuvant component (if it possesses immune-stimulatory properties) or as a model antigen for studying immune responses against iridovirus proteins. For studies examining viral inhibition of host immune responses, comparative analysis of wild-type and modified IIV6-140L variants could identify functional domains responsible for potential immune evasion activities. Additionally, since IIV6 infection has been shown to protect cells from subsequent arboviral infection, investigating whether IIV6-140L contributes to this protective effect could yield insights into novel antiviral strategies .

What are the potential applications of IIV6-140L in cross-species infection models?

IIV6-140L offers valuable research applications in cross-species infection models that examine viral host-switching and adaptation mechanisms. Researchers can develop experimental systems using IIV6-140L as a marker to track viral movement between invertebrate vectors and vertebrate hosts, particularly in reptile and amphibian models where natural infections have been documented . By creating recombinant viruses with modified 140L genes, scientists can investigate the protein's potential role in determining host range or tissue tropism across species barriers. Cell culture models incorporating both invertebrate and vertebrate cells (such as insect cells alongside reptilian or mammalian cells) can be used to examine how IIV6-140L function differs between host types and potentially contributes to successful cross-species transmission . These experimental systems are particularly relevant for understanding the ecological dynamics of iridovirus infections in environments where invertebrate vectors and vertebrate hosts coexist. Moreover, since IIV6 has been shown to stimulate mammalian immune responses despite not productively infecting mammals, the study of IIV6-140L in mammalian systems may reveal novel mechanisms of viral recognition across species boundaries .

How might IIV6-140L research contribute to understanding broader virus-host interactions?

Research on IIV6-140L has potential to advance fundamental understanding of virus-host interactions across multiple biological domains. The study of this protein exemplifies how non-mammalian viruses can interact with mammalian immune systems in unexpected ways, challenging conventional understandings of viral recognition mechanisms . By investigating the structural and functional properties of IIV6-140L, researchers may identify novel protein domains involved in host specificity determination or immune evasion that could have parallels in medically relevant viruses. The cross-reactivity between invertebrate virus components like IIV6-140L and vertebrate immune sensors provides a unique experimental system for studying the evolutionary conservation of antiviral defense mechanisms across distant phylogenetic groups. Understanding how IIV6-140L potentially contributes to the documented protective effect of IIV6 against subsequent arboviral infections could reveal new mechanisms of viral interference relevant to disease prevention strategies . Additionally, characterizing the interactome of IIV6-140L across different host species may identify conserved cellular pathways targeted by diverse viruses, potentially revealing new therapeutic targets for viral diseases. This research represents an important bridge between invertebrate virology and vertebrate immunology with broad implications for understanding viral ecology and pathogenesis.

What are the common challenges in expressing and purifying recombinant IIV6-140L?

Researchers working with recombinant IIV6-140L face several technical challenges that require specific optimization strategies. The hydrophobic nature of portions of the IIV6-140L protein sequence can lead to poor solubility and aggregation during expression and purification procedures . Expression systems selection is critical; while E. coli systems offer high yield and simplicity, the protein may form inclusion bodies requiring denaturing and refolding protocols that can compromise structural integrity. Baculovirus expression systems using insect cells may provide more appropriate post-translational modifications but at lower yields. The small size of the protein (64 amino acids) presents challenges for detection and quantification, often requiring fusion tags that may influence structural properties . Purification protocols typically require optimization of buffer conditions, with the addition of detergents or stabilizing agents to maintain solubility of hydrophobic regions. Storage stability issues are also common, necessitating careful buffer formulation (typically Tris-based with 50% glycerol) and avoidance of repeated freeze-thaw cycles . Researchers should validate the structural integrity and functionality of purified recombinant 140L using circular dichroism spectroscopy, limited proteolysis, and functional binding assays before proceeding to experimental applications.

What controls should be included in experimental designs involving IIV6-140L?

Robust experimental design for studies involving IIV6-140L requires careful consideration of appropriate controls to ensure valid and interpretable results. For immunological studies, researchers should include both positive controls (such as known immunostimulatory molecules like poly(dA:dT) for DNA sensing pathways) and negative controls (unrelated viral proteins with similar biochemical properties) to establish the specificity of immune responses to IIV6-140L . When examining protein-protein interactions, control experiments should include both "bait-only" and "prey-only" conditions to identify non-specific binding, along with unrelated proteins of similar size and charge characteristics as specificity controls. For viral infection studies comparing wild-type and 140L-mutant viruses, complementation controls (reintroducing the wild-type gene) are essential to confirm that observed phenotypes are directly attributable to 140L function rather than secondary mutations . Temperature controls are particularly important when working with IIV6, as experiments often require incubation at temperatures suitable for both invertebrate (typically 28°C) and vertebrate systems (37°C), which may independently affect various biological processes . Time-course experiments should include multiple sampling points to capture the dynamic nature of viral infection and host response, with appropriate mock-infected controls at each timepoint to account for changes in baseline cellular conditions.

What analytical methods are most appropriate for studying IIV6-140L structure-function relationships?

A comprehensive analytical toolkit is essential for elucidating the structure-function relationships of IIV6-140L. For structural characterization, circular dichroism spectroscopy provides initial insights into secondary structure composition, while nuclear magnetic resonance (NMR) spectroscopy is particularly suitable for determining the three-dimensional structure of smaller proteins like IIV6-140L (64 amino acids) . Computational approaches including molecular dynamics simulations and homology modeling based on structurally characterized viral proteins can generate testable hypotheses about functional domains. Site-directed mutagenesis targeting conserved residues identified through comparative sequence analysis, followed by functional assays, can systematically map regions critical for protein activity. For membrane association studies suggested by the protein's hydrophobic regions, fluorescence microscopy using tagged IIV6-140L variants and subcellular fractionation approaches are appropriate analytical methods. Protein-protein interaction studies should employ multiple complementary techniques, including co-immunoprecipitation, biolayer interferometry for binding kinetics, and proximity ligation assays in cellular contexts. Cross-linking mass spectrometry can identify specific contact residues between IIV6-140L and binding partners, providing detailed molecular interaction maps. Integrating these diverse analytical approaches yields a comprehensive understanding of how 140L structure relates to its biological function in the viral life cycle.

How should researchers interpret contradictory findings regarding IIV6-140L function?

When confronted with contradictory findings regarding IIV6-140L function, researchers should employ a systematic analytical framework to reconcile discrepancies. First, methodological differences between studies should be carefully evaluated, as variations in expression systems, purification protocols, and experimental conditions can significantly impact protein behavior and functionality . The biological context is equally important—results from invertebrate cell systems may differ fundamentally from those in vertebrate models due to divergent host factors and cellular environments . Researchers should consider whether contradictions reflect true biological complexity rather than experimental artifacts, as IIV6-140L may possess context-dependent multifunctionality across different hosts or cellular compartments. Statistical analysis of replicate experiments with appropriate sample sizes and power calculations can help distinguish significant effects from experimental noise. When contradictions persist despite methodological standardization, researchers should develop integrative models that incorporate seemingly disparate findings into a coherent understanding of 140L function under different conditions. Collaborative cross-laboratory validation studies using standardized reagents and protocols represent the gold standard for resolving persistent contradictions and establishing consensus on IIV6-140L function.

What is the significance of IIV6-140L in understanding virus evolution across species barriers?

The study of IIV6-140L provides a valuable model for understanding the molecular basis of viral host range and cross-species transmission events. IIV6 occupies an interesting ecological niche, with documented ability to infect both invertebrate hosts and cold-blooded vertebrates like reptiles and amphibians, suggesting proteins like 140L may play roles in overcoming species barriers . Comparative genomic analyses have identified several differences between IIV6 and related viruses, including deletions, recombination sites, and insertions of genes from non-iridoviral sources, highlighting the dynamic evolutionary processes that shape viral genomes when adapting to new hosts . The functional characterization of IIV6-140L across different host systems can reveal how viral proteins maintain, lose, or gain functions during host adaptation processes. Particularly intriguing is the observation that IIV6 can activate mammalian immune responses through the RIG-I pathway despite being a DNA virus from an invertebrate host, suggesting unexpected molecular mimicry or recognition patterns that transcend species boundaries . Understanding these dynamics has broader implications for predicting viral emergence, as the mechanisms enabling IIV6 to cross species barriers may have parallels in other viral systems with zoonotic potential.

What future research directions should be prioritized for IIV6-140L studies?

Future research on IIV6-140L should prioritize several key directions to address critical knowledge gaps. Structural biology approaches, particularly NMR spectroscopy or X-ray crystallography of the purified protein, would provide essential insights into the three-dimensional architecture underpinning its function . Comprehensive host-interaction studies using techniques such as BioID proximity labeling or protein microarrays across multiple host species would identify conserved and divergent binding partners in different biological contexts. Functional genomics approaches employing CRISPR-Cas9 to create 140L knockout or mutant viruses, followed by comparative phenotypic and transcriptomic analyses, would definitively establish the protein's role in the viral life cycle . Evolutionary analyses comparing 140L sequences across diverse iridovirus isolates from various hosts would reveal selection pressures and adaptive changes associated with host switching events. Immunological studies should further investigate whether 140L contributes to the observed ability of IIV6 to stimulate RIG-I pathway activation in mammalian cells and potential protective effects against subsequent arboviral infections . Additionally, development of high-throughput screening assays to identify small molecule modulators of 140L function could provide valuable research tools and potential antiviral lead compounds. Integration of these multidisciplinary approaches would significantly advance our understanding of this enigmatic viral protein and its biological significance.

Comparative Protein Characteristics of IIV6-140L and Related Viral Proteins

Recommended Experimental Protocols for IIV6-140L Studies

IIV6-140L Expression Systems Comparison