Recombinant Spodoptera frugiperda ascovirus 1a Uncharacterized protein ORF43 (ORF43)

What is known about the temporal expression pattern of ORF43 in SfAV-1a infection?

While specific expression data for ORF43 has not been fully characterized, transcriptome analysis of SfAV-1a reveals that viral genes can be classified into three temporal expression categories: early, late, and very late. To determine the temporal expression pattern of ORF43, researchers should employ strand-specific RNA-Seq analysis at multiple timepoints post-infection (6, 12, 24, 48 hpi, and 4-7 days post-infection), similar to the approach used for other SfAV-1a genes . The percentage of viral reads typically increases from 0.007% at 6 hpi to 3.76% by 7 days post-infection, providing a timeline for studying temporal expression .

How does the genomic location of ORF43 relate to other characterized genes in SfAV-1a?

Given that SfAV-1a gene expression shows spatial and temporal organization, researchers should map ORF43 in relation to the 44 core genes that occur across multiple ascovirus species. This mapping would help determine if ORF43 is located near genes involved in nucleotide metabolism, lipid metabolism, apoptosis regulation, structural proteins, or host interaction proteins . Researchers should be aware that SfAV-1a contains unusual bi-/tricistronic mRNA messages, so ORF43 may be co-transcribed with other genes .

What sequence homology does ORF43 share with proteins from other ascovirus species?

To address this question, researchers should conduct BLASTP analysis against other ascovirus genomes, particularly focusing on Heliothis virescens ascovirus (HvAV) and Trichoplusia ni ascovirus (TnAV). The search results indicate that some SfAV genes are conserved only in SfAV and HvAV . Initial homology searches may not yield significant matches due to the uncharacterized nature of ORF43, necessitating more sensitive approaches like position-specific iterative BLAST (PSI-BLAST) or hidden Markov model (HMM) profiling.

What experimental approaches are most effective for determining the function of ORF43 in viral vesicle formation?

Investigating ORF43's potential role in viral vesicle formation requires a multi-faceted approach:

• Temporal correlation analysis: Determine if ORF43 expression correlates temporally with vesicle formation events, similar to how caspase (ORF073) expression occurs very late, coinciding with apoptotic events leading to viral vesicle formation .

• Localization studies: Generate recombinant ORF43 tagged with fluorescent proteins (similar to the mClover3-H2B approach used in HCoV-OC43 studies ) to track its subcellular localization during infection.

• Protein-protein interaction analysis: Identify binding partners using co-immunoprecipitation or proximity labeling techniques to determine if ORF43 interacts with known vesicle formation machinery.

• Knockout/knockdown studies: Employ CRISPR-Cas9 or RNA interference to reduce ORF43 expression and observe effects on vesicle morphology and viral replication.

How might ORF43 contribute to SfAV-1a's ability to modulate host cell apoptosis?

SfAV-1a's ability to modulate host cell apoptosis involves a complex interplay between early expression of apoptosis inhibitors (IAP-like proteins; ORF016, ORF025, and ORF074) and very late expression of caspase (ORF073) . To investigate ORF43's potential role:

• Expression timing analysis: Determine if ORF43 is expressed early (suggesting a potential role in apoptosis inhibition) or late/very late (suggesting involvement in vesicle formation).

• Domain prediction: Use computational tools to identify potential anti-apoptotic or pro-apoptotic domains within ORF43.

• Apoptosis assays: Overexpress or knockdown ORF43 and measure effects on cellular markers of apoptosis (caspase activation, phosphatidylserine exposure, DNA fragmentation).

• Interaction studies: Test for direct interactions between ORF43 and known apoptotic machinery or with other SfAV-1a apoptosis modulators.

What structural characteristics of ORF43 can be predicted, and how might they inform functional hypotheses?

A comprehensive structural characterization would include:

• Secondary structure prediction: Use algorithms like PSIPRED, JPred, or SOPMA to predict α-helices, β-sheets, and disordered regions.

• Transmembrane domain analysis: Tools like TMHMM or Phobius can predict if ORF43 contains membrane-spanning regions that might localize it to viral vesicles.

• 3D structure prediction: AlphaFold2 or RoseTTAFold can generate predicted tertiary structures to identify potential functional sites.

• Motif scanning: Search for conserved functional motifs using PROSITE, PFAM, or SMART databases.

• Post-translational modification prediction: Identify potential phosphorylation, glycosylation, or other modification sites that might regulate ORF43 function.

The structural predictions should be validated experimentally using techniques such as circular dichroism spectroscopy or X-ray crystallography.

What is the optimal expression system for producing recombinant ORF43 for functional and structural studies?

Several expression systems should be considered based on research objectives:

For structural studies, researchers should consider expressing ORF43 in insect cells due to the importance of native folding and post-translational modifications. For initial characterization, E. coli systems may be sufficient. The yeast-based assembly system described for coronavirus mutagenesis could potentially be adapted for ascovirus studies to generate recombinant viruses expressing tagged ORF43.

How can RNA-Seq data be optimally processed to accurately determine ORF43 expression levels during SfAV-1a infection?

Based on the methodologies used for other SfAV-1a genes, researchers should:

• Generate strand-specific RNA-Seq libraries from infected Spodoptera frugiperda larvae at multiple timepoints (6, 12, 24, 48 hpi, and 4-7 days post-infection) .

• Map reads to the SfAV-1a genome after manual curation to correct sequence errors, as RNA-Seq has been shown to significantly improve genome reannotation .

• Quantify expression using RPKM values (Reads Per Kilobase Million), with highly expressed genes typically showing values >1,000 RPKM .

• Perform differential expression analysis to classify ORF43 into temporal expression categories (early, late, or very late) .

• Validate expression patterns using RT-qPCR with primers specific to ORF43.

• Identify potential bi-/tricistronic mRNAs containing ORF43, as SfAV-1a has been reported to have at least 15 such messages .

What techniques can be employed to determine if ORF43 plays a role in SfAV-1a's immune evasion strategies?

SfAV-1a employs several strategies to overcome host immune responses, including expression of a Diedel homolog (ORF121) and RNase III (ORF022) . To investigate ORF43's potential role in immune evasion:

• Host gene expression analysis: Compare host immune gene expression in infections with wild-type versus ORF43-mutant viruses.

• Reporter assays: Develop reporter systems that measure activation of immune signaling pathways (e.g., JAK/STAT, Toll, IMD) in the presence or absence of ORF43.

• Protein-protein interaction studies: Identify interactions between ORF43 and host immune proteins using yeast two-hybrid or co-immunoprecipitation approaches.

• Recombinant virus studies: Generate an ORF43 knockout or tagged virus using reverse genetics systems similar to those developed for other viruses , then assess changes in virulence or host response.

• Viral competition assays: Compare the replication efficiency of wild-type versus ORF43-mutant viruses in immunocompetent versus immunocompromised hosts.

How does ORF43 compare to highly expressed uncharacterized proteins in SfAV-1a?

The search results indicate that several SfAV-1a genes (ORF121, ORF107, ORF032, and ORF055) are highly expressed (>1,000 RPKMs) at certain stages of infection . Researchers should:

• Compare expression patterns between ORF43 and these highly expressed genes to identify potential functional relationships.

• Determine if ORF43 shares sequence or structural similarities with ORF032 and ORF055, which have no known function but are highly expressed .

• Analyze evolutionary conservation patterns, noting that ORF055 is conserved in all ascovirus species while ORF032 is conserved in HvAV and TnAV .

• Investigate whether ORF43 falls into any of the five main functional classes identified in ascoviruses: nucleotide metabolism, lipid metabolism, apoptosis, structural proteins, or host interaction proteins .

What insights can be gained from comparing ORF43 with functionally characterized proteins from related large DNA viruses?

To position ORF43 within the broader context of large DNA virus biology:

• Perform phylogenetic analyses comparing ORF43 with homologs from iridescent viruses and phycodnaviruses, which are more closely related to ascoviruses than baculoviruses .

• Apply comparative genomics approaches to identify conserved domains or motifs shared with functionally characterized proteins from related viruses.

• Consider functional analogies rather than just sequence homology, as proteins with similar functions may have diverged significantly at the sequence level while maintaining structural similarities.

• Investigate whether ORF43 has functional counterparts in other DNA viruses that undergo nuclear lysis and vesicle formation during their replication cycle.

What are the main technological challenges in developing a robust cell culture system for studying ORF43 function in vitro?

The search results indicate that "a robust cell line to study the novel molecular biology of ascovirus replication in vitro is lacking" . Key challenges include:

• Identifying permissive cell lines: Researchers need to systematically screen insect cell lines for their ability to support complete SfAV-1a replication, including vesicle formation.

• Establishing infection parameters: Determine optimal MOI, timing, and conditions for observing the full replication cycle.

• Developing fluorescent reporter systems: Create recombinant viruses expressing fluorescent proteins (similar to the mClover3-H2B approach used for coronaviruses ) to enable real-time monitoring of infection and protein localization.

• Replicating the in vivo environment: Design culture conditions that mimic the in vivo environment of Spodoptera frugiperda larvae to support complete viral replication.

• Creating stable reporter cell lines: Develop cell lines that express fluorescent markers for specific cellular structures to visualize the interaction of ORF43 with host components.

What future experimental approaches could elucidate the role of ORF43 in the context of the complete SfAV-1a transcriptome?

As research on SfAV-1a continues to advance, several approaches could provide deeper insights into ORF43 function:

• Single-cell RNA-Seq: Apply single-cell transcriptomics to understand cell-to-cell variability in ORF43 expression and identify cell subpopulations with distinct expression patterns.

• Ribosome profiling: Determine if ORF43 is efficiently translated and identify potential regulatory mechanisms affecting its translation.

• CRISPR-Cas9 genome editing: Develop CRISPR systems for ascovirus modification to create ORF43 knockouts, tagged variants, or domain-specific mutants.

• Cryoelectron tomography: Visualize the three-dimensional localization of ORF43 within viral vesicles and virions.

• Synthetic biology approaches: Apply the yeast-based DNA assembly techniques used for coronavirus reverse genetics to create modular systems for ascovirus manipulation and study.

• Protein-protein interaction networks: Generate comprehensive interactomes to position ORF43 within the network of viral and host protein interactions during infection.