Recombinant Ictalurid herpesvirus 1 Uncharacterized protein ORF13 (ORF13)

What is the genomic location and structure of ORF13 in Ictalurid herpesvirus 1?

ORF13 is located within the CCV genome adjacent to ORF12. These two ORFs are in the same reading frame but are separated by three stop codons that prevent read-through translation. This genomic organization suggests independent functions despite their proximity . The gene is positioned in the region following the transcriptional unit encoding ORF12, which has been characterized as a 1,412 bp transcript with its 5′ end located at nucleotide positions 15,368 and 131,043 in the CCV genome .

What structural motifs are present in the ORF13 protein?

ORF13 contains a potential zinc metal binding motif near its amino terminus, similar to the motif found in ORF12 . Zinc binding motifs are commonly involved in protein-protein interactions and in binding DNA and RNA molecules. This structural feature may suggest potential regulatory functions for ORF13, possibly in transcriptional or post-transcriptional processes, though this requires experimental verification .

How does ORF13 compare to homologous proteins in other herpesviruses?

While specific homology data for ORF13 is limited in the available literature, the zinc-binding domain structure places it in context with other herpesvirus regulatory proteins. For comparison, herpes simplex virus type 1 (HSV-1) immediate early proteins ICP0 and ICP27 contain zinc metal binding domains that contribute to their regulatory functions . A comprehensive sequence alignment analysis would be required to identify specific homologies across the Herpesviridae family.

What is known about the temporal expression pattern of ORF13?

• Time-course RNase protection assays on CCV-infected cell lysates

• Northern blot analysis using ORF13-specific probes

• Real-time PCR to quantify transcript levels at various timepoints post-infection

• Investigation of the effects of protein synthesis inhibitors (like cycloheximide) and DNA replication inhibitors (like phosphonoacetic acid) on ORF13 expression

What promoter elements control ORF13 expression?

The specific promoter elements for ORF13 have not been characterized in detail. By comparison, other CCV gene promoters show varying structures:

To characterize the ORF13 promoter, researchers would need to perform:

• 5′ RACE to identify the transcriptional start site

• Sequence analysis of the upstream region to identify potential regulatory elements

• Reporter gene assays to functionally validate promoter activity

What bacterial artificial chromosome (BAC) systems exist for generating recombinant Ictalurid herpesvirus 1 with ORF13 modifications?

The CCV genome has been successfully cloned as three overlapping subgenomic bacterial artificial chromosomes (BACs), which provides a powerful system for generating recombinant viruses including those with ORF13 modifications . The methodological approach would involve:

• Utilizing the established overlapping BAC system to target ORF13

• Employing homologous recombination in E. coli to introduce desired modifications

• Co-transfecting channel catfish ovary (CCO) cells with the modified BACs

• Isolating and characterizing the resultant recombinant viruses

This system has been validated through the successful generation of an ORF12 deletion mutant, demonstrating its utility for targeted modification of CCV genes .

What techniques are most effective for purifying recombinant ORF13 protein for functional studies?

For effective purification of recombinant ORF13, researchers should consider:

• Expression system selection:

  • Bacterial systems (E. coli) for high yield but potential folding issues

  • Eukaryotic systems (insect cells, mammalian cells) for proper folding and post-translational modifications

• Affinity tag strategy:

  • N-terminal or C-terminal His6 or GST tags to facilitate purification

  • TEV or PreScission protease cleavage sites for tag removal

• Purification protocol:

  • Initial capture using affinity chromatography

  • Secondary purification using ion exchange chromatography

  • Final polishing using size exclusion chromatography

• Consideration of the zinc-binding domain:

  • Inclusion of zinc ions in purification buffers

  • Avoiding strong chelating agents that might disrupt metal binding

How can researchers assess potential DNA/RNA binding activity of ORF13?

Given the presence of a zinc binding motif suggesting possible nucleic acid interaction, researchers should consider these methodological approaches:

• Electrophoretic Mobility Shift Assays (EMSA):

  • Using labeled DNA/RNA fragments from the CCV genome

  • Competition assays with unlabeled nucleic acids to determine specificity

• Chromatin Immunoprecipitation (ChIP):

  • In infected cells to identify genomic binding sites in vivo

  • Followed by sequencing (ChIP-seq) for genome-wide binding profile

• RNA Immunoprecipitation (RIP):

  • To identify RNA targets if the protein functions in post-transcriptional regulation

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • For quantitative binding kinetics measurements

  • Determination of binding constants for various nucleic acid targets

What experimental approaches can determine if ORF13 is essential for viral replication?

To determine whether ORF13 is essential for viral replication, researchers can employ methodologies similar to those used for ORF12 characterization:

• Construction of an ORF13 deletion mutant using the BAC system:

  • Design targeting constructs with appropriate homology arms

  • Replace ORF13 with a selection marker

  • Verify deletion by restriction enzyme analysis and sequencing

• Functional analysis of the deletion mutant:

  • Transfect CCO cells with the mutant BAC

  • Monitor for virus production and plaque formation

  • Compare growth kinetics with wild-type virus

  • Analyze viral protein expression patterns

The experimental approach established for ORF12 (which was found to be nonessential for replication) provides a validated methodology that can be adapted for ORF13 studies .

What is known about potential protein-protein interactions involving ORF13?

The specific protein interaction partners of ORF13 have not been characterized in the available literature. Given its zinc binding motif, which is often involved in protein-protein interactions, several experimental approaches would be appropriate:

• Yeast two-hybrid screening:

  • Using ORF13 as bait against a channel catfish cDNA library

  • Alternative: against a library of CCV viral proteins

• Co-immunoprecipitation (Co-IP) studies:

  • Using antibodies against ORF13 or an epitope-tagged version

  • Mass spectrometry analysis of co-precipitated proteins

• Proximity labeling approaches:

  • BioID or APEX2 fusions to ORF13

  • Expression in infected cells followed by streptavidin pulldown and mass spectrometry

• Protein microarray analysis:

  • Screening purified ORF13 against arrays of viral or host proteins

How does ORF13 potentially contribute to viral pathogenesis in channel catfish?

The role of ORF13 in CCV pathogenesis remains uncharacterized. To investigate this, researchers could employ:

• In vivo studies with ORF13 mutant viruses:

  • Infection of juvenile channel catfish with wild-type and mutant viruses

  • Monitoring of disease progression, mortality rates, and viral loads

  • Histopathological examination of infected tissues

• Ex vivo tissue culture models:

  • Infection of primary channel catfish cell types

  • Analysis of cytopathic effects and viral replication efficiency

• Host response analysis:

  • Transcriptome analysis of infected tissues

  • Cytokine profiling to assess immunomodulatory effects

  • Analysis of apoptotic pathways activation

How conserved is ORF13 across different isolates of Ictalurid herpesvirus 1?

To assess conservation of ORF13, researchers should:

• Perform sequence analysis of ORF13 from multiple CCV isolates:

  • PCR amplification and sequencing of the ORF13 region

  • Whole genome sequencing of diverse isolates

• Conduct bioinformatic analysis:

  • Multiple sequence alignment

  • Calculation of nucleotide and amino acid conservation rates

  • Identification of conserved domains versus variable regions

• Construct phylogenetic trees:

  • Based on ORF13 sequences compared to whole genome trees

  • Analysis of selection pressures using dN/dS ratios

What methodologies are appropriate for studying potential post-translational modifications of ORF13?

Given the regulatory nature of many herpesvirus proteins with zinc binding domains, ORF13 may undergo post-translational modifications. Appropriate methodologies include:

• Mass spectrometry-based approaches:

  • Immunoprecipitation of ORF13 from infected cells

  • Tryptic digestion and LC-MS/MS analysis

  • Targeted analysis for phosphorylation, SUMOylation, and ubiquitination

• Western blot analysis:

  • Using modification-specific antibodies

  • Mobility shift assays for modifications affecting protein size

• In vitro modification assays:

  • Testing recombinant ORF13 as substrate for various modifying enzymes

  • Kinase assays, SUMOylation assays, etc.

• Mutagenesis studies:

  • Site-directed mutagenesis of predicted modification sites

  • Functional comparison of mutants with wild-type protein

What are effective strategies for overcoming difficulties in generating antibodies against ORF13?

Generating specific antibodies against viral proteins like ORF13 can be challenging. Recommended approaches include:

• Epitope selection strategies:

  • Computational prediction of antigenic regions

  • Focus on hydrophilic, surface-exposed segments

  • Avoid regions with high similarity to host proteins

• Multiple immunization approaches:

  • Full-length recombinant protein

  • Synthetic peptides conjugated to carrier proteins

  • DNA immunization with ORF13 expression constructs

• Alternative detection methods:

  • Epitope tagging of recombinant viruses (HA, FLAG, etc.)

  • Generation of nanobodies or aptamers as binding reagents

• Validation strategies:

  • Testing antibody specificity using ORF13 deletion mutants

  • Preabsorption controls with recombinant protein

  • Western blot, IFA, and IP validation protocols

What technical considerations are important when designing CRISPR/Cas9 approaches for ORF13 modification in the viral genome?

CRISPR/Cas9 approaches for modifying ORF13 in the CCV genome require careful consideration:

• Guide RNA design:

  • Target unique sequences within ORF13

  • Avoid regions with similarity to host genomic sequences

  • Assess potential off-target effects using bioinformatic tools

• Delivery strategies:

  • For BAC modification in E. coli: plasmid-based expression

  • For direct viral genome editing: ribonucleoprotein complexes

• Repair template design:

  • Include appropriate homology arms (500-1000 bp)

  • Consider marker genes for selection (fluorescent proteins, antibiotic resistance)

  • Design in-frame modifications to avoid disrupting adjacent genes

• Screening and validation:

  • PCR-based screening of clones

  • Sequencing confirmation

  • Functional testing of modified viruses