Recombinant Ostreid herpesvirus 1 Uncharacterized protein ORF90 (ORF90)

What is Ostreid herpesvirus 1 (OsHV-1) and why is ORF90 significant?

Ostreid herpesvirus 1 (OsHV-1) is a major bivalve pathogen associated with severe mortality events across a wide host range, most notably in the Pacific oyster (Crassostrea gigas), an economically important aquaculture species. OsHV-1 contains multiple open reading frames (ORFs) encoding proteins with various functions, with ORF90 being one of the uncharacterized proteins within its genome . The significance of ORF90 lies in its potential role in viral pathogenicity, host specificity, or immune evasion, making it a target for researchers investigating disease resistance mechanisms in bivalves. Understanding ORF90 could contribute to breeding programs focused on developing OsHV-1 resistant oyster strains .

How does ORF90 relate to the broader genomic architecture of OsHV-1?

ORF90 is one of several uncharacterized proteins in the OsHV-1 genome. The virus has a single-stranded DNA genome that encodes multiple structural and non-structural proteins. While comprehensive genomic studies have identified significant SNPs and genomic regions associated with herpesvirus-caused mortalities (5,271 SNPs and 1,883 genomic regions covering 3,111 genes in larvae, and 18,692 SNPs and 28,314 regions covering 4,863 genes in adults), the specific role of ORF90 within this genetic landscape requires further investigation . The protein may function in concert with other viral factors to facilitate infection, replication, or immune evasion in bivalve hosts.

What experimental approaches are commonly used to express recombinant ORF90 protein?

Expression of recombinant ORF90 typically follows standard molecular biology protocols adapted for viral proteins:

• Gene synthesis or PCR amplification from viral genomic DNA

• Cloning into an appropriate expression vector with purification tags

• Transformation into expression hosts (commonly E. coli BL21(DE3) or similar strains)

• Optimization of expression conditions including:

  • Induction temperature (typically 16-30°C)

  • Inducer concentration (IPTG at 0.1-1.0 mM)

  • Expression duration (4-24 hours)

• Purification via affinity chromatography using added tags

• Quality control via SDS-PAGE and Western blotting

For challenging viral proteins like ORF90, eukaryotic expression systems such as insect cells (baculovirus) or yeast might yield better results for proper folding and potential post-translational modifications.

How can structure prediction methodologies be applied to understand ORF90 function in the absence of experimental structures?

In the absence of experimental structures, computational approaches similar to those used for other viral proteins can be applied to ORF90. The methodology would involve:

• Sequence analysis using UniProtKB and ExPASy proteomic tools to determine physicochemical parameters

• Selection of appropriate modeling approaches:

  • Comparative modeling if suitable templates exist (typically with >30% sequence identity)

  • Ab initio modeling for unique structural features

• Structure verification using quality assessment tools:

  • QMEAN for global structure evaluation

  • Ramachandran plot analysis for stereochemical quality

  • ERRAT for non-bonded interaction patterns

For example, comparable studies with other viral proteins achieved verification scores of 94-95% residues in favored regions of Ramachandran plots and ERRAT quality factors of 97-100, indicating high reliability of theoretical structures . Structural predictions can then inform functional hypotheses, particularly regarding potential interaction sites with host proteins or nucleic acids.

What is the relationship between ORF90 polymorphisms and OsHV-1 resistance in different developmental stages of Pacific oysters?

While specific data on ORF90 polymorphisms is limited, research on OsHV-1 broadly indicates that antiviral response or resistance mechanisms differ between larvae and adults. Pooled whole-genome resequencing revealed that only 1,653 implicated genes were shared between larvae and adults, suggesting developmental stage-specific defense mechanisms . For ORF90, this raises important questions:

• Does ORF90 show differential expression or functional significance at different developmental stages?

• Are polymorphisms in ORF90 or its regulatory regions associated with resistance?

• Does the protein interact differently with host immune components in larvae versus adults?

To investigate these questions, researchers would need to conduct comparative analyses of ORF90 sequence variations between susceptible and resistant oyster populations, coupled with transcriptomic and functional studies across developmental stages.

How do variants of ORF90 differ across OsHV-1 strains isolated from different host species?

OsHV-1 has been documented in various bivalve species, including a variant (OsHV-1-SB) associated with mortalities in blood clam (Scapharca broughtonii) broodstocks . A comprehensive analysis of ORF90 variations across different OsHV-1 strains would involve:

• Genomic sequencing of OsHV-1 variants from different hosts

• Multiple sequence alignment of ORF90 coding sequences

• Identification of conserved and variable regions

• Correlation of variations with host range and virulence

Such analysis could reveal host-specific adaptations and potentially identify regions of ORF90 under selective pressure, providing insights into its functional role in host-pathogen interactions.

What are the optimal conditions for analyzing ORF90 interactions with host immune receptors?

Based on research with other OsHV-1 proteins and host immune interactions, investigating ORF90's potential interactions with host immune receptors would involve:

Given the importance of pattern recognition receptors (PRRs) like TLRs and RLRs in oyster antiviral responses, these would be priority candidates for interaction studies with ORF90 . The choice of methodology should consider the practical constraints of working with marine invertebrate immune components, including the availability of specific antibodies and recombinant proteins.

How can transcriptional regulation of ORF90 be studied during OsHV-1 infection cycles?

Investigating ORF90 transcriptional regulation would require:

• Time-course sampling following controlled OsHV-1 infection

• RNA extraction and quality control

• Transcript quantification using:

  • RT-qPCR for targeted analysis

  • RNA-Seq for genome-wide expression patterns

• Promoter analysis to identify regulatory elements:

  • Computational prediction of transcription factor binding sites

  • Chromatin immunoprecipitation (ChIP) to confirm binding

  • Reporter assays to validate functional significance

Similar approaches with other OsHV-1 genes revealed that antiviral pattern recognition receptor genes (TLRs and RLRs) show significant transcriptional upregulation following infection, with important transcription factors including IRF, NF-kappa B, HSF, C/EBP, and AP-1 . Analyzing ORF90 in this context could provide insights into its regulation during the viral replication cycle.

How should researchers interpret contradictory findings about ORF90 functions across different experimental systems?

When confronted with contradictory findings about ORF90 functions, researchers should:

• Evaluate methodological differences:

  • Expression systems used (prokaryotic vs. eukaryotic)

  • Host cell types or organisms studied

  • Experimental conditions (temperature, salinity, etc.)

• Consider biological factors:

  • Developmental stage of host organisms

  • Genetic background of hosts (resistant vs. susceptible strains)

  • Viral strain variations

• Apply statistical rigorous analysis:

  • Assess statistical power of contradictory studies

  • Examine effect sizes rather than just p-values

  • Consider meta-analysis approaches when multiple studies exist

• Design validation experiments:

  • Use orthogonal experimental approaches

  • Include appropriate controls

  • Replicate key findings under standardized conditions

Understanding that antiviral responses differ between developmental stages (as shown by the limited overlap of implicated genes between larvae and adults) provides context for potentially contradictory findings about ORF90 functions.

What bioinformatic approaches best identify potential functional domains in ORF90?

To identify potential functional domains in ORF90, researchers should employ:

• Sequence-based analyses:

  • BLAST and PSI-BLAST for identifying distant homologs

  • Multiple sequence alignment to identify conserved residues

  • Motif scanning using PROSITE, Pfam, and SMART databases

  • Disorder prediction to identify structured vs. unstructured regions

• Structure-based predictions:

  • Secondary structure prediction

  • Tertiary structure modeling (comparative or ab initio)

  • Functional site prediction (binding pockets, catalytic sites)

  • Molecular dynamics simulations to assess flexibility

• Integration with experimental data:

  • Mapping of known mutations affecting function

  • Correlation with transcriptomic responses

  • Proteomics data on post-translational modifications

Similar approaches applied to other viral proteins have successfully identified functional elements, such as tunnels with specific dimensions (bottleneck radius, length, throughput) that may be involved in substrate binding or catalysis .

How can researchers distinguish between direct and indirect effects of ORF90 on host immune responses?

Distinguishing direct from indirect effects of ORF90 on host immune responses requires:

• Direct interaction studies:

  • Purified component binding assays

  • Co-localization in infected cells

  • CRISPR-mediated knockout or mutation studies

• Temporal analysis:

  • Time-course studies to establish sequence of events

  • Pulse-chase experiments to track protein dynamics

  • Conditional expression systems for temporal control

• Pathway dissection approaches:

  • Selective inhibition of specific signaling pathways

  • Phosphoproteomics to identify signal transduction changes

  • Genetic knockdowns of pathway components

• Systems biology integration:

  • Network analysis of transcriptomic/proteomic data

  • Mathematical modeling of immune response kinetics

  • Correlation of ORF90 activities with specific immune signatures

Research on other OsHV-1 components has revealed that variations in regulatory regions of immune genes like TLRs and RLRs are associated with resistance, suggesting transcriptional regulation as a key mechanism . Similar approaches could determine whether ORF90 directly modulates these pathways or acts through other mechanisms.

What are the most promising approaches for developing ORF90-based strategies to improve OsHV-1 resistance in aquaculture?

Based on current understanding of OsHV-1 resistance mechanisms, promising approaches include:

• Genetic marker development:

  • Identification of SNPs associated with ORF90 recognition/response

  • Development of high-throughput screening methods

  • Implementation in marker-assisted selection breeding programs

• Functional validation studies:

  • CRISPR-based genome editing to modify host receptors

  • Development of peptide inhibitors targeting ORF90

  • Creation of attenuated viral strains with modified ORF90

• Comparative genomics:

  • Analysis of ORF90 variants across resistant and susceptible populations

  • Multi-species comparisons to identify conserved resistance mechanisms

  • Integration with existing genomic data on OsHV-1 resistance

Previous research identified 5,271 SNPs and 1,883 genomic regions covering 3,111 genes in larvae, and 18,692 SNPs and 28,314 regions in adults associated with herpesvirus resistance . Focusing on those potentially interacting with ORF90 could accelerate the development of resistance strategies.

How might structural information about ORF90 inform the design of antiviral interventions?

Structural information about ORF90 would inform antiviral interventions through:

• Structure-based drug design:

  • Identification of potential binding pockets

  • Virtual screening of compound libraries

  • Fragment-based drug discovery approaches

• Peptide inhibitor development:

  • Design of peptides mimicking interaction interfaces

  • Optimization for stability and cellular uptake

  • Testing in cell culture and animal models

• Antibody development:

  • Identification of surface-exposed epitopes

  • Rational design of neutralizing antibodies

  • Engineering for improved affinity and specificity

• Vaccine design strategies:

  • Identification of immunogenic regions

  • Development of subunit or epitope-based vaccines

  • Assessment of protective efficacy in challenge studies

Similar structure prediction approaches for other viral proteins have identified features such as tunnels with specific dimensions (bottleneck radius of 1.2-1.9 Å, lengths of 1.5-5.8 Å) that could serve as targets for small molecule inhibitors .