Recombinant Diadromus pulchellus idnoreovirus 1 Uncharacterized protein S10 (S10)

What is Diadromus pulchellus Idnoreovirus 1 and its relationship to other reoviruses?

Diadromus pulchellus Idnoreovirus 1 (DpIRV1) is a species from the genus Idnoreovirus within the family Reoviridae. Phylogenetic comparisons have identified relationships between Seg-2 and Seg-10 of related reoviruses and genes of DpIRV1 . Unlike cypoviruses which have a single-layered capsid structure, idnoreoviruses like DpIRV1 have a double-layered capsid structure . The host organism, Diadromus pulchellus, is an endoparasitic wasp in the family Ichneumonidae that has been established in Northeastern United States and Canada since its first appearance in Ontario in 1993 .

How is the DpIRV1 S10 gene typically characterized in laboratory settings?

Characterization of the DpIRV1 S10 gene typically involves molecular cloning and sequence analysis. Researchers isolate viral RNA, perform reverse transcription, and amplify the S10 gene using PCR with specific primers targeting conserved regions. Based on methodologies used for related viruses, the complete open reading frame (ORF) of the S10 segment can be amplified using designed oligonucleotides . The PCR product is then purified, ligated into a vector (such as pGEM-T), and transformed into E. coli for sequence verification and further analysis . Sequence analysis should focus on identifying conserved terminal motifs characteristic of reoviruses and predicting the functional domains of the encoded protein.

What expression systems are most effective for producing recombinant DpIRV1 S10 protein for structural and functional studies?

The baculovirus expression system has proven effective for similar reovirus proteins and would be the recommended approach for DpIRV1 S10 protein. Based on protocols used for related viral proteins, the procedure involves:

• Amplification of the complete S10 ORF using designed primers

• Cloning into a transfer vector such as pBacPAK8 using compatible restriction sites

• Co-transfection of Spodoptera frugiperda (Sf-21) cells with the recombinant transfer vector and baculovirus DNA

• Amplification of recombinant virus and protein expression analysis

SDS-PAGE analysis can confirm successful expression, with expected protein size around 54 kDa based on similar reovirus S10 proteins . Western blotting with specific antibodies can further verify protein identity. Unlike cypovirus polyhedrins, expression of reovirus S10 proteins typically does not result in the formation of occlusion bodies in infected cells .

How can researchers distinguish between DpIRV1 S10 and other related idnoreovirus proteins in phylogenetic analyses?

Phylogenetic distinction of DpIRV1 S10 from other idnoreovirus proteins requires:

• Multiple sequence alignment of the complete S10 amino acid sequences using MUSCLE or CLUSTALW algorithms

• Construction of phylogenetic trees using maximum likelihood or Bayesian methods

• Validation through bootstrap analysis (1000+ replicates)

• Comparison of conserved motifs specific to each viral genus

The RNA-dependent RNA polymerase (RdRp) sequence, encoded by a different segment but highly conserved, serves as the gold standard for reovirus classification and should be included in analyses when available . While segment-specific phylogenies may show incongruence due to reassortment events, researchers should look for unique sequence signatures in the S10 protein that differentiate DpIRV1 from related viruses. Specifically, analysis of terminal sequences can be valuable, as conserved motifs such as 5′-AAATAAA...G/TAGGTT-3′ are found in most reovirus genome segments, with exceptions like 5′-AACAAA...-3′ in some segments .

What are the methodological challenges in studying recombination events involving DpIRV1 S10?

Studying recombination events involving DpIRV1 S10 presents several methodological challenges:

• Detection of true recombination versus sequencing artifacts or chimeric sequences generated during PCR

• Distinguishing between recombination and reassortment events in segmented RNA viruses

• Limited availability of complete sequence data for idnoreoviruses

• Determining recombination breakpoints with precision

Researchers should employ multiple recombination detection methods including:

• RDP4 software suite (implementing RDP, GENECONV, Bootscan, MaxChi)

• Split decomposition analysis

• Phylogenetic incongruence testing

• Breakpoint analysis using GARD or similar algorithms

Norovirus recombination studies provide methodological precedents, as they have identified 80 intergenotypic recombinant types, including intergenogroup recombinants . Similar to noroviruses, researchers should investigate both field isolates and laboratory conditions that might favor recombination in idnoreoviruses .

How does the amino acid composition of DpIRV1 S10 protein compare to polyhedrin proteins in cypoviruses, and what are the functional implications?

The amino acid composition of DpIRV1 S10 protein differs significantly from cypovirus polyhedrins in several key aspects:

These compositional differences explain why DpIRV1 S10 protein does not form occlusion bodies like cypovirus polyhedrins . The high leucine content suggests potential involvement in protein-protein interactions or signaling functions that might contribute to viral pathogenesis through cell necrosis-inducing activities . Hydrophobicity plotting and secondary structure prediction algorithms show distinct patterns compared to polyhedrins, further supporting functional divergence .

What are the recommended protocols for isolating and purifying recombinant DpIRV1 S10 protein for antibody production?

To isolate and purify recombinant DpIRV1 S10 protein for antibody production, researchers should follow this methodological workflow:

• Expression optimization:

  • Express the complete S10 ORF in a baculovirus expression system using Sf-21 cells

  • Optimize expression conditions (MOI, harvest time, temperature)

  • Confirm expression via SDS-PAGE (expected size ~54 kDa)

• Protein purification:

  • Lyse infected cells in buffer containing appropriate detergents and protease inhibitors

  • Clarify lysate by centrifugation (10,000×g, 30 min, 4°C)

  • Purify using affinity chromatography (if His-tagged) or ion exchange chromatography

  • Further purify by size exclusion chromatography to ensure homogeneity

  • Verify purity by SDS-PAGE and Western blotting

• Antibody production:

  • Immunize rabbits with 250-500 μg purified protein in complete Freund's adjuvant

  • Boost with 100-250 μg protein in incomplete Freund's adjuvant at 2-3 week intervals

  • Collect sera and purify IgG using Protein A/G

  • Validate antibody specificity using Western blotting against both recombinant protein and virus-infected samples

This methodology ensures production of specific antibodies that can be used for immunolocalization studies, protein quantification, and functional assays.

What bioinformatic approaches are most effective for predicting functional domains and potential binding partners of DpIRV1 S10?

Effective bioinformatic prediction of DpIRV1 S10 functional domains requires a multi-layered analysis approach:

• Primary sequence analysis:

  • Search for conserved domains using NCBI CDD, Pfam, SMART, and InterPro

  • Identify functional motifs using ELM, PROSITE, and ScanProsite

  • Analyze for signal peptides and transmembrane regions using SignalP and TMHMM

• Structural prediction:

  • Generate secondary structure predictions using PSIPRED, JPred

  • Perform tertiary structure modeling using AlphaFold2 or I-TASSER

  • Validate models using PROCHECK, VERIFY3D, and ProSA

• Interaction prediction:

  • Use STRING, STITCH, and PrePPI for protein-protein interaction prediction

  • Identify potential RNA-binding regions using RBPmap and catRAPID

  • Analyze protein surface electrostatics using APBS to identify potential binding sites

• Evolutionary analysis:

  • Conduct evolutionary trace analysis to identify functionally important residues, similar to approaches used for norovirus capsid proteins

  • Map trace residues onto structural models to visualize functional regions

Additionally, researchers should perform comparative analyses with related viral proteins where functional data exists, particularly focusing on leucine-rich regions that may be involved in signaling motifs relevant to cell necrosis-inducing activities .

How can researchers effectively design CRISPR-Cas9 systems to study the function of DpIRV1 S10 in host-pathogen interactions?

Designing CRISPR-Cas9 systems to study DpIRV1 S10 function requires strategic approaches tailored to RNA viruses:

• Target selection and gRNA design:

  • Analyze the S10 sequence for highly conserved regions using multiple sequence alignments

  • Design 3-5 gRNAs targeting different regions of the S10 segment

  • Assess gRNAs for off-target effects using tools like Cas-OFFinder or CRISPOR

  • Design gRNAs with NGG PAM sites and optimal GC content (40-60%)

• Delivery system optimization:

  • For in vitro studies, use lentiviral vectors expressing Cas9 and gRNAs

  • For in vivo studies in Diadromus pulchellus, consider microinjection of Cas9-gRNA ribonucleoproteins

  • Include appropriate selection markers and reporters (GFP, mCherry)

• Validation strategies:

  • Confirm editing efficiency using T7E1 assay, Surveyor assay, or deep sequencing

  • Verify target specificity using whole-genome sequencing

  • Quantify viral replication using qRT-PCR targeting other viral segments

• Functional analysis:

  • Perform transmission electron microscopy to assess virion formation

  • Conduct host gene expression analysis using RNA-seq

  • Analyze virus-host protein interactions using co-immunoprecipitation followed by mass spectrometry

This methodological framework enables researchers to precisely target the S10 segment, leading to functional insights regarding its role in viral replication, host range determination, and pathogenesis.

What role might the DpIRV1 S10 protein play in determining host specificity and cross-species transmission potential?

The DpIRV1 S10 protein may contribute significantly to host specificity and cross-species transmission potential. The relatively high leucine content (10.04%) in related reovirus S10 proteins suggests potential involvement in signaling pathways that could influence host cell interactions . When investigating this role, researchers should consider:

• Comparative studies between S10 proteins from reoviruses with different host ranges

• Identification of host cellular receptors or interaction partners through pull-down assays and mass spectrometry

• Recombinant virus studies with chimeric S10 segments to assess changes in host range

Researchers should explore whether the S10 protein functions similarly to norovirus recombinants, where recombination events have been linked to zoonotic potential . Since Diadromus pulchellus is an endoparasitic wasp that primarily targets leek moth (Acrolepiopsis assectella) , studies should examine whether the S10 protein facilitates viral adaptation between the wasp host and its insect prey, potentially allowing for cross-species transmission.

How might structural studies of DpIRV1 S10 contribute to our understanding of reovirus evolution?

Structural studies of DpIRV1 S10 would significantly enhance our understanding of reovirus evolution by:

• Providing insights into structural adaptations that differentiate idnoreoviruses from cypoviruses, particularly regarding the absence of occlusion body formation capabilities

• Identifying conserved structural elements across different reovirus genera that may represent ancestral features

• Revealing structural motifs that could be involved in host adaptation and cross-species transmission

• Elucidating the structural basis for segmented genome organization and packaging

These studies should employ:

• Cryo-electron microscopy to determine high-resolution structures

• X-ray crystallography of purified S10 protein

• Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Molecular dynamics simulations to investigate conformational changes

Researchers could draw methodological parallels from evolutionary trace analysis used in norovirus studies, which has successfully identified capsid protein residues that uniquely characterize different norovirus strains . Such approaches would reveal whether similar evolutionary patterns exist in idnoreoviruses and how the S10 protein contributes to viral adaptation.

What are the most promising applications of recombinant DpIRV1 S10 protein in developing novel biocontrol strategies?

The recombinant DpIRV1 S10 protein holds potential for developing novel biocontrol strategies, particularly because its host organism, Diadromus pulchellus, is already established as an effective parasitoid of leek moth (Acrolepiopsis assectella) . Research applications include:

• Developing virus-based biocontrol agents that could enhance the efficacy of D. pulchellus as a parasitoid

• Engineering recombinant viruses with modified S10 proteins to increase specificity against target pests

• Creating diagnostic tools to monitor viral prevalence in parasitoid populations