Recombinant Invertebrate iridescent virus 6 Uncharacterized protein 113L (IIV6-113L)

What is IIV6-113L and where is it encoded in the viral genome?

IIV6-113L is one of the 215 non-overlapping and putative protein-encoding open reading frames (ORFs) found in the genome of Invertebrate iridescent virus 6 (IIV6), the type species of the Iridovirus genus within the Betairidovirinae subfamily of the Iridoviridae family . The IIV6 genome consists of 212,482 bp of linear double-stranded DNA that encodes these various proteins with different functions in the viral life cycle . The designation "113L" indicates both the numerical position of this ORF within the genome and the orientation of transcription (with "L" denoting the leftward direction), following the standard nomenclature system used for iridovirus genes . Despite being identified as a distinct ORF, IIV6-113L remains largely uncharacterized in terms of its specific function, unlike some other IIV6 proteins such as the 118L envelope protein that has been conclusively shown to be essential for viral replication . Researchers working with IIV6-113L typically use recombinant versions of the protein for functional studies, as evidenced by commercially available preparations like the His-tagged recombinant full-length protein expressed in E. coli systems .

To which temporal expression class does IIV6-113L belong?

The transcription of IIV6 genes is temporally regulated, dividing them into three kinetic classes: immediate-early (IE), delayed-early (DE), and late (L) . Recent comprehensive RT-PCR analysis of total RNA isolated from virus-infected insect cells, conducted in the presence or absence of protein and DNA synthesis inhibitors, has classified all 215 viral ORFs including previously uncharacterized ones . While the search results don't explicitly state the temporal class of IIV6-113L specifically, this methodology has successfully placed 113 genes in the IE class, 23 in the DE class, and 22 in the L class . The temporal classification of viral genes provides important insights into their potential roles during the infection cycle, with IE genes typically involved in regulatory functions, DE genes in DNA replication, and L genes in virion assembly and structural components . To definitively determine the expression class of IIV6-113L, researchers can replicate the established experimental approach using protein synthesis inhibitor cycloheximide (to identify IE genes) and DNA synthesis inhibitor aphidicolin (to identify DE genes), while genes expressed only in the absence of both inhibitors would be classified as L genes . Understanding the temporal expression pattern of IIV6-113L would provide valuable clues about its potential function in the viral replication cycle.

What conserved motifs might regulate IIV6-113L expression?

The expression of IIV6 genes is regulated by specific conserved motifs in their upstream regions that vary according to their temporal classification . For immediate-early (IE) genes, the conserved motif AA(A/T)(T/A)TG(A/G)A has been identified in the upstream region and validated through luciferase reporter assays as crucial for promoter activity . Delayed-early (DE) genes contain the conserved motif (T/A/C)(T/G/C)T(T/A)ATGG in their upstream regions, which has similarly been confirmed as essential for their expression . Late (L) genes have a more complex regulatory pattern with two initially identified conserved 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) . Interestingly, rather than enhancing expression, these motifs were found to act as repressors, with the second motif containing highly conserved nucleotides at the end (TTGT) that function specifically as a repressor sequence . After removing this repressor sequence in silico, researchers identified two additional motifs crucial for L gene expression: (T/A)(A/T)(A/T/G)(A/T)(T/C)(A/G)(A/C)(A/C) and (C/G)(T/C)(T/A/C)C(A/T)(A/T)T(T/G)(T/G)(T/G/A), with mutations in either causing severe decreases in expression . To determine which motifs might regulate IIV6-113L, researchers would need to first identify its temporal class and then analyze its upstream region (approximately 200 bp) for the presence of these class-specific conserved sequences.

What are the most effective methods for expressing and purifying recombinant IIV6-113L for structural studies?

For effective expression and purification of recombinant IIV6-113L, researchers should consider established protocols that have been successful with other IIV6 proteins. Based on the available data, bacterial expression systems, particularly E. coli, have been employed successfully for producing recombinant IIV6 proteins, as evidenced by the commercially available His-tagged recombinant IIV6-113L . When designing expression constructs, researchers should consider adding affinity tags such as polyhistidine (His) tags to facilitate purification through immobilized metal affinity chromatography (IMAC) . For structural studies requiring properly folded proteins, insect cell expression systems might be more appropriate, as they have been successfully used for virus protein analysis in IIV6 research . The purification strategy should include multiple chromatography steps, potentially combining IMAC with size exclusion chromatography to achieve high purity levels necessary for structural studies. Mass spectrometry approaches, such as LC-ESI LTQ Orbitrap MS/MS or LC-MALDI TOF/TOF MS, which have been successfully applied to other IIV6 proteins, can be employed to verify the identity and purity of the recombinant protein . If solubility issues arise during expression, researchers might need to optimize buffer conditions or consider expressing truncated versions of the protein based on predicted domain structures. Additionally, when studying viral membrane proteins, detergent screening might be necessary to maintain protein stability during purification, although the available data doesn't explicitly indicate whether IIV6-113L is a membrane protein.

How can researchers determine if IIV6-113L is a structural component of the virion?

To determine if IIV6-113L is a structural component of the IIV6 virion, researchers should employ a comprehensive proteomic approach similar to what has been used successfully for other iridovirus structural proteins . The first critical step involves the purification of IIV6 virions using established protocols such as density gradient ultracentrifugation to ensure high purity . The purified virions should then be denatured in SDS-PAGE sample buffer, with proteins separated on 10% SDS-PAGE gels following standard techniques . For comprehensive protein identification, researchers should employ complementary mass spectrometry approaches, such as both LC-MALDI TOF/TOF MS and LC-ESI LTQ Orbitrap MS/MS, which have been successfully used in previous IIV6 virion proteome analyses . The mass spectrometry data should be processed using appropriate software (such as 4000 series Explorer software for MALDI TOF/TOF data and Proteome Discoverer for ESI LTQ Orbitrap data) and searched against databases containing all predicted IIV6 ORFs . As a control comparison, proteins from IIV6-infected insect cells can be analyzed in parallel to distinguish virion structural proteins from non-structural viral proteins that are expressed in infected cells but not incorporated into mature virions . Additional confirmation could come from immunoelectron microscopy using antibodies raised against recombinant IIV6-113L to visualize the potential localization of the protein within virion structures. Western blot analysis of purified virions versus infected cell lysates collected at different time points post-infection would provide further evidence of the protein's structural or non-structural nature.

What experimental approaches would best determine IIV6-113L's role in viral replication?

To determine IIV6-113L's role in viral replication, researchers should employ multiple complementary experimental approaches that have proven successful with other IIV6 proteins. Gene deletion through homologous recombination, replacing the target gene with a reporter gene such as GFP, allows for visual tracking of mutant viruses and has been effectively used with other IIV6 proteins such as 118L . If the mutant virus cannot be propagated independently of wild-type virus, this suggests the protein may be essential for viral replication, as observed with IIV6-118L . RNA interference using double-stranded RNA (dsRNA) targeting the IIV6-113L gene represents another powerful approach, with successful precedent in IIV6 research where a 99% reduction in virus titer was achieved following dsRNA treatment targeting essential genes . Researchers should also consider developing antibodies against recombinant IIV6-113L to conduct neutralization assays, determining if antibody treatment can inhibit virus infection, which would suggest a role in virus entry or early replication events . Complementary to these approaches, researchers could employ time-course studies using quantitative PCR and Western blotting to track expression patterns of IIV6-113L during infection, correlating these with specific phases of the viral replication cycle. Protein-protein interaction studies using techniques such as co-immunoprecipitation or yeast two-hybrid assays could identify viral or host proteins that interact with IIV6-113L, providing further clues to its function. Subcellular localization studies using fluorescently tagged versions of the protein would determine which cellular compartments contain IIV6-113L during infection, offering additional insights into its potential role.

What bioinformatic tools are most useful for predicting IIV6-113L protein structure and function?

For comprehensive bioinformatic analysis of the uncharacterized IIV6-113L protein, researchers should employ multiple complementary tools and approaches. Sequence analysis should begin with BLASTP against protein databases to identify homologs in other viruses, particularly within the Iridoviridae family, which can provide initial functional insights based on evolutionary conservation patterns . For protein domain and motif prediction, InterProScan is particularly valuable as it has been successfully applied in previous IIV6 studies, integrating multiple databases to identify conserved domains that might suggest function . Transmembrane domain prediction using tools like SignalP and PredictProtein can determine if IIV6-113L contains membrane-spanning regions similar to the essential envelope protein 118L, which has three predicted transmembrane domains . For structural analysis in the absence of experimental data, modern protein structure prediction tools such as AlphaFold2 or RoseTTAFold can generate reliable structural models that may reveal functional features not apparent from sequence analysis alone. To identify potential post-translational modifications, researchers should analyze the sequence for N-glycosylation and N-myristoylation sites, which have been found to be important in other IIV6 proteins like 118L . Repetitive elements within the protein sequence can be identified using specialized tools like XSTREAM, with subsequent alignments of protein repeats generated using alignment software like MegAlign . The temporal expression pattern of IIV6-113L can be inferred by analyzing its upstream regulatory region (200 bp) for the presence of conserved promoter motifs identified for IE, DE, or L genes using motif discovery tools such as the MEME Suite Software, which has been previously applied to IIV6 genome analysis .

How can researchers develop effective antibodies against IIV6-113L for immunological studies?

Developing effective antibodies against IIV6-113L requires a strategic approach that begins with antigen preparation. Researchers should express and purify recombinant full-length IIV6-113L protein with affinity tags, such as the His-tagged version available commercially, ensuring high purity for immunization . If the full-length protein presents solubility challenges, researchers can alternatively identify immunogenic epitopes through computational prediction and synthesize corresponding peptides conjugated to carrier proteins like KLH (Keyhole Limpet Hemocyanin) for immunization. For polyclonal antibody production, researchers should immunize rabbits or other suitable animals with the purified recombinant protein or peptide conjugates following established immunization schedules with appropriate adjuvants, collecting sera at different time points to monitor antibody titers. For monoclonal antibody development, researchers should consider using BALB/c mice for immunization, followed by hybridoma technology where B cells from immunized mice are fused with myeloma cells to create immortalized antibody-producing cell lines. Antibody specificity must be rigorously validated through multiple complementary approaches: Western blot analysis against both recombinant IIV6-113L and IIV6-infected cell lysates to confirm recognition of the native protein; immunoprecipitation to verify antibody-antigen binding under native conditions; immunofluorescence microscopy to assess subcellular localization in infected cells; and cross-reactivity testing against other IIV6 proteins to ensure specificity. Functional assays should be performed to determine if the antibodies possess neutralizing activity against IIV6 infection, similar to the neutralizing antibodies developed against IIV6-118L, which successfully neutralized virus infection . For advanced applications, researchers might consider developing antibody fragments like Fab or scFv which may offer advantages for certain experimental approaches requiring smaller immunological reagents.

What cell culture systems and infection protocols yield optimal results for studying IIV6-113L expression during viral infection?

For optimal study of IIV6-113L expression during viral infection, researchers should select appropriate insect cell lines that support productive IIV6 infection. Based on the search results, Sf21 cells (derived from Spodoptera frugiperda) have been successfully used for IIV6 infection studies and represent a primary choice for investigating viral protein expression . When establishing infection protocols, researchers should standardize the multiplicity of infection (MOI) based on experimental objectives, with higher MOIs (5-10 PFU/cell) recommended for synchronous infection when studying early expression events and lower MOIs (0.1-1 PFU/cell) for studying the complete viral replication cycle. Time-course experiments should be designed to capture the full temporal progression of infection, with sampling points typically ranging from 0 to 72 hours post-infection, with more frequent sampling during early infection phases to precisely determine when IIV6-113L expression begins . For studying the temporal class of IIV6-113L expression, researchers should employ selective inhibitors as used in previous IIV6 studies: cycloheximide (100 μg/ml) to block protein synthesis for identifying immediate-early genes, and aphidicolin (5 μg/ml) to inhibit DNA synthesis for differentiating between delayed-early and late genes . RNA extraction protocols should be optimized for viral transcripts, with RT-PCR or quantitative RT-PCR employing primers specific to IIV6-113L, designed to span exon-exon junctions if applicable to avoid genomic DNA amplification . For protein detection, both Western blot analysis using antibodies against IIV6-113L and mass spectrometry approaches such as LC-ESI LTQ Orbitrap MS/MS should be employed for comprehensive protein identification and quantification throughout the infection cycle . Additionally, researchers might consider developing fluorescently tagged versions of IIV6-113L for real-time visualization of protein expression and localization during infection through live-cell imaging techniques.

How does IIV6-113L compare with homologous proteins in other members of the Iridoviridae family?

Comparing IIV6-113L with homologous proteins in other members of the Iridoviridae family requires a systematic approach combining bioinformatic analysis with experimental validation. Researchers should begin with comprehensive BLASTP searches against all sequenced iridovirus genomes to identify potential homologs based on sequence similarity . Unlike IIV6-118L, which is known to be conserved across all sequenced members of the Iridoviridae family, the conservation pattern of IIV6-113L across the family is not explicitly stated in the available search results . Once homologs are identified, multiple sequence alignment using tools like MUSCLE or Clustal Omega can reveal conserved residues and domains that may indicate functionally important regions of the protein. Phylogenetic analysis of these aligned sequences can provide insights into the evolutionary relationships between the homologs and potentially correlate with host range or virulence characteristics of different iridoviruses. Structural comparison of the predicted protein structures, using tools like AlphaFold2 for modeling, may reveal conserved structural features even in cases where sequence conservation is limited. Researchers should also compare the genomic context of IIV6-113L homologs across different iridoviruses, as conserved gene arrangements (synteny) can provide additional evidence for homology and functional relationships. For experimental validation, researchers can perform cross-complementation assays where the IIV6-113L homolog from another iridovirus is expressed in IIV6-infected cells where the native 113L has been knocked down or deleted, to determine if functional complementation occurs. Additionally, generating antibodies against IIV6-113L and testing cross-reactivity with homologous proteins from other iridoviruses can provide insights into structural conservation that might not be apparent from sequence analysis alone.

How do the promoter elements of IIV6-113L compare with those of other temporal classes in the IIV6 genome?

The comparative analysis of IIV6-113L promoter elements with those of other temporal classes requires a detailed examination of upstream regulatory sequences in the context of established IIV6 transcriptional patterns. In the IIV6 genome, gene expression is temporally regulated with distinct promoter motifs identified for each kinetic class . For immediate-early (IE) genes, the conserved motif AA(A/T)(T/A)TG(A/G)A has been validated as crucial for promoter activity through luciferase reporter assays . In contrast, delayed-early (DE) genes contain the motif (T/A/C)(T/G/C)T(T/A)ATGG in their upstream regions, which is essential for their expression . Late (L) genes exhibit a more complex regulatory pattern with identified repressor sequences including (C/G/T)(G/A/C)(T/A)(T/G)(G/T)(T/C) with highly conserved TTGT nucleotides, and activator motifs (T/A)(A/T)(A/T/G)(A/T)(T/C)(A/G)(A/C)(A/C) and (C/G)(T/C)(T/A/C)C(A/T)(A/T)T(T/G)(T/G)(T/G/A) that are essential for expression . To compare IIV6-113L with these established patterns, researchers should extract 200 bp of sequence upstream of the 113L start codon and analyze it using motif discovery tools such as the MEME Suite Software that has been successfully applied in previous IIV6 promoter studies . The presence or absence of these class-specific motifs would provide initial insights into the likely temporal class of IIV6-113L. For experimental validation, researchers could clone the 113L upstream region into a luciferase reporter construct and perform reporter assays in infected cells, comparing the expression pattern with known IE, DE, and L gene promoters under various conditions, including in the presence of protein or DNA synthesis inhibitors . Additionally, introducing targeted mutations in any identified conserved motifs through site-directed mutagenesis would confirm their functional significance in regulating IIV6-113L expression.

How might codon optimization strategies differ when expressing IIV6-113L in different host systems?

Codon optimization strategies for expressing IIV6-113L require tailored approaches depending on the host expression system, as codon usage preferences vary significantly between organisms. When expressing IIV6-113L in E. coli, which has already been successfully done as evidenced by the commercially available His-tagged recombinant protein, researchers should optimize for E. coli's distinct codon bias that heavily favors codons ending in G or C . For expression in insect cell systems, which might provide more native-like post-translational modifications for a viral protein, codon optimization should target the specific host insect cell line being used, such as Sf21 (Spodoptera frugiperda) which has been successfully employed in IIV6 research . When designing synthetic genes, researchers should consider not only codon adaptation index (CAI) but also address potential issues such as mRNA secondary structures, cryptic splice sites, and sequence repetitions that might impair expression efficiency regardless of codon optimality. Different optimization algorithms may yield varying results, so researchers should compare outputs from multiple optimization tools and potentially test several variants experimentally to identify the optimal sequence for their specific expression system. For membrane proteins or those with complex folding requirements, more conservative optimization approaches that maintain some rare codons, particularly at domain boundaries, might be beneficial for proper folding kinetics. Researchers should also consider the presence of any regulatory elements within the coding sequence that might be disrupted by optimization, although this is less likely to be an issue with viral proteins being expressed in heterologous systems. Finally, when expressing IIV6-113L in mammalian cells for applications such as antibody production or protein-protein interaction studies, optimization should account for the distinct GC content requirements and codon preferences of mammalian systems, while avoiding sequences that might trigger innate immune responses such as CpG motifs.

What CRISPR-based approaches could be applied to study IIV6-113L function?

CRISPR-based technologies offer powerful approaches for studying IIV6-113L function, although their application to viral genomes requires specialized strategies. For direct editing of the IIV6 genome, researchers could design CRISPR-Cas9 systems targeting the 113L locus, with guide RNAs (gRNAs) designed using algorithms that maximize on-target efficiency while minimizing off-target effects in both the viral and host genomes . Complete knockout of IIV6-113L could be achieved by introducing frameshifts or premature stop codons, while more subtle functional analysis could utilize CRISPR-mediated homology-directed repair to introduce specific mutations in predicted functional domains or to add reporter tags like GFP for visualization . The successful approach used to replace IIV6-118L with GFP through homologous recombination provides a methodological template that could be adapted to CRISPR-based strategies for 113L . For temporal regulation studies, CRISPR interference (CRISPRi) using catalytically inactive Cas9 (dCas9) fused to transcriptional repressors could be employed to suppress 113L expression at specific time points during infection, potentially providing insights into its temporal requirement during the viral life cycle . Conversely, CRISPR activation (CRISPRa) using dCas9 fused to transcriptional activators could be used to upregulate 113L expression, potentially overriding its normal temporal regulation. For studying host factor interactions, CRISPR screens in susceptible insect cell lines could identify host genes that, when knocked out, alter the phenotype of IIV6-113L mutants or affect 113L protein function, potentially revealing cellular pathways involved in 113L's role during infection. When implementing these approaches, researchers should establish appropriate controls including non-targeting gRNAs and validate editing efficiency through sequencing, while monitoring for potential confounding effects such as large deletions or chromosomal rearrangements that can occur with CRISPR-Cas9 systems.

How can researchers design experiments to identify host protein interactions with IIV6-113L?

To comprehensively identify host protein interactions with IIV6-113L, researchers should employ multiple complementary approaches that capture different types of protein-protein interactions. Affinity purification coupled with mass spectrometry (AP-MS) represents a powerful initial approach, where researchers can express tagged versions of IIV6-113L (such as His-tagged or FLAG-tagged constructs) in insect cells, perform pull-downs under various conditions, and identify co-purifying host proteins using LC-ESI LTQ Orbitrap MS/MS or similar high-sensitivity mass spectrometry techniques . Proximity-based labeling methods such as BioID or APEX2, where IIV6-113L is fused to a biotin ligase or peroxidase that biotinylates nearby proteins, can capture transient or weak interactions that might be lost during conventional co-immunoprecipitation procedures. Yeast two-hybrid screening using IIV6-113L as bait against insect cell cDNA libraries can identify direct binary interactions, though this approach requires careful validation due to potential false positives. For more physiologically relevant interaction studies, researchers should conduct experiments in the context of viral infection, comparing the interactome of IIV6-113L at different time points post-infection to identify temporal changes in host factor interactions that might correspond to different stages of the viral life cycle . Computational approaches can complement experimental methods, with protein-protein interaction prediction algorithms suggesting potential host binding partners based on structural compatibility and previously characterized virus-host protein interactions. All potential interactions identified through these approaches should be validated through orthogonal methods such as co-immunoprecipitation, bimolecular fluorescence complementation (BiFC), or Förster resonance energy transfer (FRET) to confirm their specificity and relevance. Functional validation of key interactions can be achieved through siRNA knockdown or CRISPR knockout of identified host factors, assessing the impact on viral replication and specifically on IIV6-113L localization or function.

What strategies can be employed to determine if IIV6-113L undergoes post-translational modifications?

To comprehensively investigate potential post-translational modifications (PTMs) of IIV6-113L, researchers should employ multiple complementary techniques targeting different types of modifications. Mass spectrometry-based approaches, particularly LC-ESI LTQ Orbitrap MS/MS, represent the gold standard for PTM identification, as they can detect and localize modifications with high sensitivity and accuracy . For these analyses, researchers should purify IIV6-113L from both recombinant sources and virus-infected cells, using enrichment strategies specific to the PTM of interest (e.g., phosphopeptide enrichment for phosphorylation, lectin affinity for glycosylation). Based on the knowledge that other IIV6 proteins like 118L contain predicted N-glycosylation and N-myristoylation sites, researchers should specifically investigate these modifications in IIV6-113L . Glycosylation can be assessed through mobility shift assays following treatment with glycosidases like PNGase F (for N-linked glycans) or O-glycosidase (for O-linked glycans), with changes in apparent molecular weight on SDS-PAGE indicating the presence of glycan structures. For lipid modifications such as myristoylation, metabolic labeling with alkyne-tagged lipid analogs followed by click chemistry and fluorescent detection can visualize these modifications with high specificity. Phosphorylation can be initially screened using phospho-specific staining techniques like Pro-Q Diamond phosphoprotein gel stain, followed by more detailed phosphosite mapping using mass spectrometry. To determine the functional significance of identified PTMs, researchers should generate site-directed mutants where the modified residues are replaced with non-modifiable amino acids (e.g., serine to alanine for phosphorylation sites) and assess the impact on protein localization, stability, and function during viral infection. Time-course studies examining when PTMs occur during the viral life cycle can provide insights into their regulatory roles, particularly in relation to the temporal expression pattern of IIV6-113L . Additionally, identifying the host enzymes responsible for these modifications through inhibitor studies or targeted knockdowns could reveal potential targets for antiviral intervention.

What are the most promising research directions for understanding IIV6-113L function?

Based on current knowledge and methodological capabilities, several research directions hold significant promise for elucidating IIV6-113L function in the viral life cycle. Temporal expression classification through RT-PCR analysis with protein and DNA synthesis inhibitors represents a critical first step, as determining whether IIV6-113L belongs to the immediate-early, delayed-early, or late gene classes would provide fundamental insights into its potential role during infection . Once classified, analyzing the upstream regulatory regions for class-specific conserved motifs using tools like MEME Suite would enhance our understanding of its transcriptional regulation mechanisms . Essential nature assessment through gene deletion or silencing approaches, similar to those successfully employed for IIV6-118L, would determine if IIV6-113L is required for viral replication and provide directions for further functional characterization . Structural biology approaches to determine the three-dimensional structure of IIV6-113L through X-ray crystallography or cryo-electron microscopy could reveal functional domains and potential interaction surfaces not identifiable through sequence analysis alone. Protein-protein interaction studies focusing on both viral and host protein partners would place IIV6-113L within the context of virus-host interaction networks and potentially reveal its role in counteracting host defenses or co-opting cellular machinery. Comparative analysis with homologs in other iridoviruses could highlight evolutionarily conserved features and potentially correlate structural differences with host range or virulence variations among iridoviruses. Cross-disciplinary approaches integrating computational predictions with experimental validation using modern techniques like CRISPR-Cas9 genome editing and high-resolution imaging would provide comprehensive insights into IIV6-113L function. Understanding this currently uncharacterized protein may reveal novel aspects of iridovirus biology with potential applications in insect pest control or development of viral vectors for gene delivery.