Recombinant Ostreid herpesvirus 1 Putative RING finger protein ORF97 (ORF97)

What is Ostreid herpesvirus 1 and what role does ORF97 play in its lifecycle?

Ostreid herpesvirus 1 (OsHV-1) is a major viral pathogen causing significant mortalities in Pacific oysters (Crassostrea gigas), particularly during early life stages. OsHV-1 infections have been reported globally, with mortality rates varying significantly between oyster families (ranging from 1.2% to 49%), suggesting a strong genetic component to disease resistance. The virus has become increasingly problematic for oyster aquaculture over the past two decades .

ORF97 encodes a putative RING finger protein of 181 amino acids with a molecular mass of 20.85 kDa . While the exact function of ORF97 in the OsHV-1 lifecycle remains to be fully characterized, its classification as a RING finger protein suggests potential E3 ubiquitin ligase activity. By analogy with other herpesvirus RING finger proteins, ORF97 may be involved in modifying host cellular processes to facilitate viral replication or evade host immune responses.

What are the structural characteristics of the ORF97 protein?

ORF97 is characterized as a putative RING finger protein from Ostreid herpesvirus 1 (isolate France). The protein consists of 181 amino acids with a molecular mass of 20.85 kDa . The complete amino acid sequence is:

MSTVKIISEEMCVACFDENVKLYIYDCGHKCCCKECFERVERCPMCRHLPVENIKPSTHVPAIPSAPSFELINMEVETREIHPRSSRAKSIAKRMVYGMGPSFLDKLFDCKSGRDSMAWGKMCCICTKKTNDEMIKSKICESYYCRDCRDYLFQENGADQPLPFCPCCFKKYNGFEHVSAH

The RING finger domain is characterized by a specific pattern of cysteine and histidine residues that coordinate zinc ions in a cross-brace structure. Based on the sequence, multiple cysteine-rich regions can be identified, particularly the motifs "CVACF" and "CCCKE" near the N-terminus, which are consistent with RING finger domain characteristics.

How does ORF97 compare to RING finger proteins in other herpesviruses?

While direct comparative data for OsHV-1 ORF97 is limited in the literature, parallels can be drawn with other herpesvirus RING finger proteins. The Varicella-Zoster virus (VZV) ORF61p, for example, contains a RING finger domain that has been extensively studied . Research has demonstrated that this domain in VZV ORF61p is necessary for E3 ubiquitin ligase activity, autoubiquitination, and regulation of protein stability. Disruption of the RING finger domain in VZV ORF61p through amino acid substitution (Cys19Gly) alters the protein's intranuclear distribution and abolishes its ability to disperse Sp100-containing nuclear bodies .

By analogy, OsHV-1 ORF97 likely shares similar functional characteristics, potentially targeting host proteins for ubiquitination and subsequent degradation as part of the viral strategy to modulate host cellular processes. The conservation of RING finger domains across diverse herpesviruses underscores their importance in viral lifecycles.

What methods are most effective for expressing and purifying recombinant ORF97?

Effective expression and purification of recombinant ORF97 requires careful consideration of expression systems and purification strategies. Based on established protocols for similar viral proteins, the following approaches are recommended:

Expression Systems:

• Prokaryotic expression: Cloning the ORF97 coding sequence into bacterial expression vectors (e.g., pET series) with appropriate tags (His-tag, GST-tag) for purification. E. coli strains BL21(DE3) or Rosetta(DE3) are commonly used for expression of herpesvirus proteins.

• Eukaryotic expression: For maintaining post-translational modifications and proper folding, baculovirus expression systems in insect cells (Sf9, Sf21, or High Five) may be preferable.

Purification Strategy:

• Affinity chromatography using the fusion tag (His-tag or GST-tag)

• Ion exchange chromatography for further purification

• Size exclusion chromatography to remove aggregates and ensure homogeneity

Critical Considerations:

• Optimizing expression conditions (temperature, induction time, inducer concentration) to balance yield and solubility

• Adding zinc supplements to the growth media and purification buffers to ensure proper folding of the RING finger domain

• Including reducing agents (DTT or β-mercaptoethanol) in buffers to maintain cysteine residues in reduced state

How can researchers assess the E3 ubiquitin ligase activity of ORF97?

Based on studies of other herpesvirus RING finger proteins, such as VZV ORF61p, ORF97 likely possesses E3 ubiquitin ligase activity . This activity can be assessed using the following approaches:

In vitro Ubiquitination Assays:

• Incubate purified recombinant ORF97 with:

  • Ubiquitin

  • E1 (ubiquitin-activating enzyme)

  • E2 (ubiquitin-conjugating enzyme, multiple E2s should be tested)

  • ATP and Mg²⁺

  • Potential substrate proteins (if known)

• Analyze reaction products by SDS-PAGE followed by Western blotting with anti-ubiquitin antibodies

Autoubiquitination Assays:
Since RING finger E3 ligases often undergo autoubiquitination, this can serve as a proxy for activity:

• Perform the in vitro assay as above, but without additional substrate

• Detect ORF97 autoubiquitination using anti-ORF97 or anti-tag antibodies

Cell-Based Assays:

• Express ORF97 in appropriate cell lines along with tagged ubiquitin

• Immunoprecipitate ORF97 or potential substrates

• Analyze ubiquitination status by Western blotting

Controls:

• RING finger mutants (e.g., mutations in conserved Cys residues) should be included as negative controls

• Known E3 ligases can serve as positive controls

• Reactions without ATP or E1/E2 enzymes provide additional negative controls

What model systems are available for studying ORF97 function in the context of OsHV-1 infection?

Studying OsHV-1 and its proteins in relevant contexts has been challenging due to the complexity of host-pathogen dynamics and lack of established laboratory models. Recent advancements have created several options:

Tissue Explant Model:
Recent research has developed tissue explant approaches for studying OsHV-1 in Pacific oysters . This model:

• Uses tissue explants taken from Pacific oysters maintained in controlled laboratory conditions

• Allows for controlled exposure to OsHV-1

• Enables monitoring of viral replication via quantitative PCR and electron microscopy

• Permits comparison between oysters from different sources with different susceptibility levels

• Provides better control of confounding factors compared to whole-animal experiments

Whole-Animal Infection Models:
Traditional approaches using laboratory infection of Pacific oysters:

• Allow for investigation of OsHV-1 pathogenesis in the natural host

• Can be used to monitor mortality rates, viral loads, and host responses

• Real-time PCR can quantify OsHV-1 DNA in both dead and living oysters during infection

• Different oyster families show significant variation in susceptibility (1.2% to 49% mortality) , allowing for comparative studies

Cell Culture Approaches:
While limited by the lack of established oyster cell lines permissive for OsHV-1 replication:

• Heterologous expression of ORF97 in mammalian or insect cells

• Co-expression with potential interaction partners

• Subcellular localization studies using fluorescently tagged ORF97

How does ORF97 expression and regulation impact OsHV-1 replication?

Understanding the regulation of ORF97 expression and its impact on viral replication remains an active area of investigation. While direct data on ORF97 regulation in OsHV-1 is limited, insights can be drawn from related systems:

Regulatory Factors:
Studies of related viral systems suggest that the expression of genes like ORF97 may be dependent on other viral proteins. For example, in AcMNPV (a baculovirus), knockout of the pp31 gene reduced transcripts from ORF97 to approximately 22% of normal levels . This suggests that ORF97 expression in OsHV-1 may similarly be regulated by other viral factors.

Impact on Viral Replication:
The contribution of ORF97 to viral replication likely involves:

• Modulation of host antiviral responses through targeted ubiquitination and degradation of host factors

• Potential regulation of viral protein stability and function

• Possible involvement in viral DNA replication or packaging through interactions with cellular or viral proteins

What are the potential host protein targets of ORF97's presumed E3 ubiquitin ligase activity?

Based on studies of RING finger proteins in other herpesviruses, ORF97 likely targets specific host proteins for ubiquitination and subsequent proteasomal degradation. While the specific targets of ORF97 have not been identified in the available literature, potential candidates include:

Immune Response Factors:

• Components of the interferon pathway

• Pattern recognition receptors

• Transcription factors involved in antiviral gene expression

Cell Cycle Regulators:

• Proteins involved in cell cycle arrest

• DNA damage response factors

• Apoptosis mediators

Chromatin-Associated Factors:
The VZV ORF61p RING finger protein has been shown to disperse Sp100-containing nuclear bodies , suggesting that ORF97 might similarly target chromatin-associated proteins involved in transcriptional regulation or intrinsic immunity.

Methodological Approaches for Target Identification:

• Proteomic analysis of ubiquitinated proteins in the presence vs. absence of ORF97

• Yeast two-hybrid screens to identify direct interaction partners

• Immunoprecipitation followed by mass spectrometry

• Candidate-based approaches focusing on known targets of other herpesvirus RING finger proteins

How do mutations in the RING finger domain affect ORF97 function and OsHV-1 pathogenesis?

The RING finger domain is critical for the function of proteins like ORF97. Based on studies of other herpesvirus RING finger proteins, mutations in this domain would likely have significant functional consequences:

Evidence from related systems supports these predictions. In Varicella-Zoster virus, a Cys19Gly substitution in the ORF61p RING finger domain alters the protein's intranuclear distribution and abolishes its ability to disperse Sp100-containing nuclear bodies. This mutation also eliminates E3 ubiquitin ligase activity and affects protein stability .

For OsHV-1, creating recombinant viruses with mutations in the ORF97 RING finger domain would allow assessment of:

• Viral replication efficiency in tissue explant models

• Changes in host gene expression patterns

• Altered pathogenesis in experimental infections

• Impact on viral gene expression programs

How should researchers quantify OsHV-1 viral load and ORF97 expression in experimental samples?

Accurate quantification of viral load and gene expression is critical for studying OsHV-1 and ORF97. The following methods have been validated and are recommended:

Viral Load Quantification:
Real-time PCR has been successfully employed to quantify OsHV-1 DNA in infected oysters . This approach:

• Allows precise measurement of viral DNA copies

• Can be applied to both dead and living individuals

• Enables tracking of infection progression

ORF97 Expression Analysis:
For specific quantification of ORF97 expression:

• RT-qPCR targeting the ORF97 transcript

• RNA-Seq for transcriptome-wide analysis

• Protein-level detection using specific antibodies against ORF97

Quantitative Data from Previous Studies:
The following table summarizes viral load data from a study of OsHV-1 infection in different oyster families:

This data demonstrates significant variation in viral loads between different oyster families, with the Z*AC family showing particularly high viral loads correlated with high mortality .

What factors influence variability in OsHV-1 infection outcomes and potentially ORF97 function?

Experimental and field studies have identified several factors that contribute to variability in OsHV-1 infection outcomes, which may influence ORF97 expression and function:

Host Genetic Factors:

• Different oyster families show dramatically different susceptibility to OsHV-1 infection

• Cumulative mortality ranges from 1.2% to 49% between families under identical exposure conditions

• Mean viral load in dead individuals varies significantly between families (P<0.001)

• The correlation between family-level mean infection and mortality suggests a genetic basis for resistance

Viral Factors:

• Potential genetic variation in ORF97 sequence between viral isolates

• Interactions with other viral proteins that may regulate ORF97 expression or function

• Viral load differences, with higher viral loads potentially leading to increased expression of viral proteins including ORF97

Environmental Factors:

• Temperature fluctuations, which can influence viral replication rates

• Water quality parameters that may stress host animals

• Seasonal variation in disease outbreaks

Methodological Considerations:
When designing experiments to study ORF97, researchers should:

• Use multiple oyster families with known susceptibility profiles

• Control environmental conditions rigorously

• Implement appropriate statistical approaches to account for biological variability

• Consider temporal dynamics of infection when sampling

How can transcriptomic data enhance our understanding of ORF97 function?

Transcriptomic approaches offer powerful insights into the role of ORF97 in OsHV-1 infection. Analysis of gene expression data can reveal:

Viral Gene Expression Patterns:

• Temporal expression profile of ORF97 during infection

• Co-expression relationships with other viral genes

• Regulatory dependencies, such as the relationship observed in other viral systems where knockout of pp31 reduced ORF97 transcript levels to approximately 22% of normal

Host Response Signatures:

• Identification of host pathways modulated during infection

• Correlation between ORF97 expression levels and host gene expression patterns

• Potential targets of ORF97's presumed E3 ubiquitin ligase activity

Comparative Transcriptomics Approaches:

• Contrast gene expression profiles between susceptible and resistant oyster families

• Compare responses to wild-type virus versus ORF97 mutants (when available)

• Analyze temporal changes in gene expression during disease progression

The power of transcriptomic approaches has been demonstrated in studies using microarray analysis of viral gene expression, which identified ORF97 as being significantly affected by knockout of regulatory proteins in related viral systems .

What approaches show promise for developing targeted interventions against OsHV-1 based on ORF97 biology?

Understanding ORF97's structure and function could lead to novel intervention strategies against OsHV-1 infection:

Small Molecule Inhibitors:

• Target the RING finger domain to disrupt E3 ubiquitin ligase activity

• Design compounds that interfere with ORF97-substrate interactions

• Develop zinc chelators that specifically disrupt RING finger structure

Genetic Selection Strategies:

• The high genetic basis underlying resistance to OsHV-1 infection suggests potential for selective breeding

• Identify genetic markers associated with reduced viral replication or ORF97 activity

• Develop oyster lines with enhanced resistance to OsHV-1

RNA Interference Approaches:

• Design siRNAs targeting ORF97 transcripts

• Develop delivery methods for RNA-based therapeutics in aquaculture settings

Vaccine Development:

• Explore the potential of recombinant ORF97 as a vaccine component

• Investigate attenuated OsHV-1 strains with modifications to ORF97

How might novel experimental systems advance ORF97 research?

Advances in experimental approaches offer new opportunities for studying ORF97:

Tissue Explant Models:
Recent development of tissue explant approaches for studying OsHV-1 provides significant advantages:

• Allows controlled infection studies outside the whole animal

• Enables precise manipulation of experimental conditions

• Facilitates comparison between tissues from oysters with different genetic backgrounds

• Permits visualization of viral replication through electron microscopy

• Reduces experimental variability compared to whole-animal studies

CRISPR/Cas9 Technology:

• Generate OsHV-1 mutants with specific modifications to ORF97

• Introduce tags for tracking ORF97 localization and interactions

• Create knock-in mutations to study the effects of specific amino acid changes

Structural Biology Approaches:

• Determine the three-dimensional structure of ORF97, particularly the RING finger domain

• Use structure-guided approaches to identify critical residues for function

• Investigate ORF97-substrate complexes to understand target recognition

What are the ecological and evolutionary implications of ORF97 research for understanding OsHV-1 adaptation?

Understanding ORF97 in an evolutionary context has broader implications for comprehending OsHV-1 adaptation and host-pathogen coevolution:

Viral Adaptation Mechanisms:

• Investigate potential sequence variation in ORF97 across viral isolates from different geographic regions

• Assess whether changes in ORF97 correlate with shifts in host range or virulence

• Examine how ORF97 sequence variation might affect substrate specificity

Host-Pathogen Coevolution:

• The significant variation in susceptibility between oyster families (1.2% to 49% mortality) suggests strong selection pressure

• Research whether host proteins targeted by ORF97 show signatures of adaptive evolution

• Investigate potential host countermeasures against ORF97 activity

Ecological Implications:

• Assess how ORF97 function might contribute to the spread and maintenance of OsHV-1 in wild and farmed oyster populations

• Investigate whether environmental factors influence ORF97 expression or activity

• Consider how selective breeding for OsHV-1 resistance might affect population genetic diversity