Recombinant Heliothis virescens ascovirus 3e Uncharacterized protein ORF18 (ORF18)

How does ORF18 protein differ from similar proteins in other ascovirus variants?

While research specifically on HvAV-3e ORF18 is limited, comparison with related ascovirus variants provides valuable insights. The Heliothis virescens ascovirus family includes several variants such as HvAV-3e, HvAV-3h, and HvAV-3i, each with distinctive genetic characteristics despite considerable homology.

For example, HvAV-3h contains a gene called 3h-31 encoding a non-structural protein (3H-31) that functions to inhibit host larval chitinase and cathepsin activities, thereby restraining hosts in their larval stages . This mechanism prevents pupation and extends the period during which the virus can replicate in the host. Although ORF18 from HvAV-3e has not been extensively characterized like 3H-31, comparative genomic analysis suggests it may have evolved similar or complementary functions given their evolutionary relationship.

Different ascovirus variants also show distinct host specificities and virulence characteristics. For instance, HvAV-3i causes mortality rates exceeding 80% in H. armigera larvae through pin inoculation, while HvAV-3h shows even higher mortality in S. exigua larvae .

What is the recommended protocol for recombinant expression of HvAV-3e ORF18 protein?

The recommended protocol for expression of recombinant ORF18 protein utilizes E. coli expression systems with a His-tag fusion to facilitate purification. The full-length ORF18 gene (encoding amino acids 1-297) is typically cloned into an expression vector with an N-terminal His-tag . Based on methodologies employed for similar ascovirus proteins, researchers should:

• Design primers incorporating appropriate restriction sites (such as BamHI and XhoI) for directional cloning of the full ORF18 sequence

• Clone the amplified gene into a suitable expression vector containing a His-tag sequence

• Transform the construct into a compatible E. coli strain optimized for protein expression

• Induce protein expression under controlled conditions (temperature, inducer concentration, duration)

• Harvest cells and extract the recombinant protein through cell lysis

• Purify using nickel affinity chromatography that specifically binds the His-tagged protein

• Assess protein purity using SDS-PAGE (target purity >90%)

The optimal expression conditions may require optimization, but generally involve induction at mid-log phase followed by expression at reduced temperatures (16-25°C) to maximize soluble protein yield.

What are the critical considerations for storage and handling of purified ORF18 protein?

For optimal stability and activity of the purified ORF18 protein, the following handling protocols are essential:

For reconstitution, the lyophilized protein should be briefly centrifuged prior to opening, then dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL . The reconstituted protein should be aliquoted to avoid repeated freeze-thaw cycles, which significantly degrade protein integrity. The buffering system typically consists of Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

How can researchers design effective experiments to investigate the function of ORF18?

To systematically investigate ORF18 function, researchers should employ multi-faceted approaches that examine both in vitro and in vivo aspects:

• Temporal expression profiling: Determine when ORF18 is expressed during viral infection using RT-PCR and Western blot analysis. Based on studies with related proteins like 3H-31, early expression (starting around 3 hours post-infection) may indicate involvement in early infection processes .

• Subcellular localization: Use fluorescent tagging (GFP fusion constructs) to track the protein's location within infected cells, which can provide insights into its functional context.

• Protein-protein interaction studies: Employ co-immunoprecipitation, yeast two-hybrid, or proximity ligation assays to identify viral or host proteins that interact with ORF18.

• Gene knockdown experiments: Utilize RNA interference (RNAi) to suppress ORF18 expression in infected larvae and observe resulting phenotypes. The dsRNA should target a unique region of ORF18 (similar to the approach used for 3h-31 gene, which targeted nucleotides 17-555) .

• Heterologous expression studies: Express ORF18 in other viral systems (such as baculovirus, similar to the AcMNPV-31 system used for 3H-31) to observe effects on viral replication, virulence, and host response .

• Host enzyme activity assays: Measure activities of key host enzymes like chitinase and cathepsin following infection with ORF18-expressing constructs, as these have been shown to be affected by related ascovirus proteins .

A comprehensive experimental design would incorporate positive and negative controls, appropriate statistical analyses, and replicate experiments to ensure reliability of results.

What are the appropriate cellular and in vivo models for studying ORF18 function?

Based on established research with related ascoviruses, the following model systems are recommended:

Cellular Models:

• SeFB cells (derived from S. exigua fat body) - previously demonstrated suitable for HvAV-3h studies

• Ha-E cells (derived from H. armigera embryo) - appropriate for studying host-virus interactions in a relevant lepidopteran system

• Sf9 or Sf21 cells - commonly used insect cell lines for recombinant protein expression

In Vivo Models:

• Spodoptera exigua (beet armyworm) larvae - shown to be susceptible to ascovirus infection and suitable for functional studies

• Helicoverpa armigera (cotton bollworm) larvae - demonstrated 80% mortality when infected with HvAV-3i, making it a relevant model for virulence studies

• Heliothis virescens (tobacco budworm) - the natural host for HvAV-3e

When conducting in vivo experiments, third-instar larvae are typically used for consistency, and larvae should be maintained at 27°C ± 1°C with a 16-h light/8-h dark photoperiod on artificial diets to standardize experimental conditions .

How should RNA interference be implemented to study ORF18 function in infected larvae?

RNA interference provides a powerful approach for functional analysis of ORF18 in vivo. Based on methodologies applied to similar ascovirus proteins, the following protocol is recommended:

• dsRNA synthesis: Generate double-stranded RNA targeting a specific region of ORF18 using in vitro transcription systems such as the T7 Ribomax Express RNAi System . The target region should be 300-600 bp in length and unique to ORF18 to avoid off-target effects.

• Control design: Prepare non-specific dsRNA (such as those targeting GFP) as negative controls to account for non-specific effects of dsRNA introduction .

• Administration: Inject 1 μg of dsRNA (concentration 1 μg/μL) directly into the hemocoel of ascovirus-infected lepidopteran larvae approximately 72 hours post-infection, when viral gene expression is established .

• Validation: Collect samples 24 hours post-dsRNA injection to confirm knockdown efficiency through RT-PCR and Western blot analysis.

• Phenotypic analysis: Monitor and quantify the following parameters:

  • Larval mortality rates

  • Developmental progression/arrest

  • Viral replication (qPCR)

  • Host enzyme activities (particularly chitinase and cathepsin)

  • Histopathological changes in larval tissues

How can transmission electron microscopy contribute to understanding ORF18 function?

Transmission electron microscopy (TEM) is invaluable for investigating the structural impact of ORF18 on viral morphogenesis and host cell ultrastructure. Based on studies with related ascovirus proteins, researchers should:

• Infect susceptible cell lines (SeFB or Ha-E) with wild-type virus and ORF18-knockout or ORF18-overexpressing variants

• Collect cells at various timepoints post-infection (typically 24, 48, and 72 hours)

• Process samples through:

  • Fixation with 2.5% glutaraldehyde (overnight)

  • Washing with PBS

  • Secondary fixation with osmium tetroxide

  • Dehydration through an ethanol series

  • Embedding in epoxy resin

  • Ultrathin sectioning (60-90 nm)

  • Staining with uranyl acetate and lead citrate

• Examine using TEM at 80 kV accelerating voltage

For related ascovirus proteins like 3H-31, TEM analysis revealed distinctive features such as lucent tubular structures around the virogenic stroma in infected cells . Similar structural studies of ORF18 may reveal its role in virion assembly, vesicle formation, or other aspects of the viral replication cycle. Quantitative analysis of virion production, vesicle dimensions, and subcellular distribution patterns should complement the qualitative observations.

How might ORF18 contribute to the development of biological control strategies?

ORF18 may hold significant potential for biological control applications based on the properties of related ascovirus proteins and the ecological role of ascoviruses:

• Enhanced virulence: Ascoviruses like HvAV-3i cause high mortality rates (>80%) in agricultural pests such as H. armigera through pin inoculation . Understanding ORF18's role in viral pathogenicity could lead to engineered virus strains with optimized pest control properties.

• Host range modification: Comparative studies of HvAV variants show different host specificities. HvAV-3i is effective against H. armigera while HvAV-3h shows higher efficacy against S. exigua . Characterizing ORF18's contribution to host specificity could enable development of broader-spectrum biopesticides.

• Integration with parasitoid wasp strategies: Ascoviruses are naturally transmitted by parasitic wasps during oviposition, creating potential for synergistic biocontrol strategies that combine viral and parasitoid approaches . ORF18 modifications might enhance virus persistence or transmission in this natural vector system.

• Developmental arrest mechanisms: If ORF18 functions similarly to 3H-31 by inhibiting chitinase and cathepsin activities, it would effectively prevent host pupation, extending the larval stage where feeding damage occurs . This mechanism offers a distinct advantage over conventional insecticides by specifically targeting development rather than directly causing mortality.

• Recombinant baculovirus systems: Engineering ORF18 into established baculovirus expression systems (similar to AcMNPV-31) could create novel bioinsecticides with enhanced properties . The delayed liquefaction phenotype observed with 3H-31 expression might improve field persistence of biopesticides.

Extensive field testing and rigorous biosafety assessment would be essential before implementation of any ORF18-based biological control strategy.

What are the evolutionary implications of ORF18 conservation across ascovirus variants?

The conservation of genes like ORF18 across ascovirus variants provides valuable insights into viral evolution and host-pathogen coevolution:

• Core viral functions: Highly conserved viral genes typically perform essential functions in viral replication or host interaction. Comparative genomic analysis of ORF18 across HvAV-3e, HvAV-3h, and HvAV-3i would reveal selection pressures acting on this gene.

• Host adaptation mechanisms: Variations in ORF18 sequences between ascovirus strains may reflect adaptations to different lepidopteran hosts. For instance, HvAV-3i causes high mortality in H. armigera, while HvAV-3h is more effective against S. exigua . These host range differences may be partly attributable to variations in ORF18 and similar genes.

• Virus-host arms race: If ORF18 functions similarly to 3H-31 in manipulating host enzyme systems (chitinase and cathepsin), it represents a sophisticated adaptation to exploit host physiology . The gene's evolution would reflect ongoing selection pressure from host counter-adaptations.

• Horizontal gene transfer potential: The relationship between ORF18 and genes from other virus families or host insects could reveal instances of horizontal gene transfer, providing insights into the evolutionary history of ascoviruses.

• Structural vs. functional conservation: Comparing the sequence conservation of different ORF18 domains across variants would highlight which regions are structurally critical (highly conserved) versus those that may be involved in host-specific interactions (more variable).

Phylogenetic analysis combining ORF18 sequences with ecological data on host range and geographical distribution would provide a comprehensive view of how this gene has evolved in response to ecological factors and host availability.