Recombinant Ostreid herpesvirus 1 Uncharacterized protein ORF71 (ORF71)

What genomic and sequence characteristics define ORF71 in the OsHV-1 genome?

ORF71 is positioned adjacent to the better-characterized ORF72 in the OsHV-1 genome. While specific ORF71 sequence characteristics are not directly mentioned in the available literature, researchers should approach its characterization through comparative genomic analysis with other OsHV-1 variants. The OsHV-1 genome contains 199,354 bp nucleotides with 38.5% G/C content, with a total of 123 ORFs putatively encoding functional proteins .

For ORF71 characterization, implement the following methodological approach:

How does ORF71 expression vary during different stages of OsHV-1 infection?

Based on transcriptomic studies of OsHV-1, viral gene expression follows temporal patterns similar to other herpesviruses. To investigate ORF71 expression:

• Use quantitative PCR targeting ORF71 transcripts at different time points post-infection (0h, 6h, 18h, 24h)

• Apply long-read transcriptomics approaches such as Nanopore DRS (Direct RNA Sequencing) to identify the complete transcript structure

• Compare expression patterns with those of adjacent genes (e.g., ORF72) to determine if they belong to the same transcriptional unit

• Determine if ORF71 undergoes alternative transcription start/stop sites or is part of polycistronic transcripts

Research indicates that viral transcripts become detectable as early as 6 hours post-incubation in hemolymph studies, with significant increases by 18 hours post-incubation, as observed with ORF72, ORF75, and ORF87 .

What computational methods are most appropriate for predicting ORF71 function?

For uncharacterized viral proteins like ORF71, employ these approaches:

• Perform homology-based function prediction using HHpred or Phyre2

• Apply machine learning-based function prediction tools (DeepFold, AlphaFold)

• Conduct comparative analysis with other Herpesvirales proteins to identify conserved functional motifs

• Employ protein-protein interaction prediction to identify potential host targets

• Use coevolution analysis to detect functionally linked proteins within the viral genome

What expression systems are optimal for producing recombinant ORF71 protein?

Based on methodologies used for other OsHV-1 proteins:

For OsHV-1 membrane proteins, the methodology used for producing recombinant proteins for antibody production has involved:

• Cloning the partial cDNA in pET-43.1a vectors

• Expressing with His-tag in N-terminal position

• Purifying using affinity chromatography

How can antibodies against ORF71 be developed and validated for research applications?

Following the approach used for ORFs 25, 41, and 72:

• Express and purify recombinant ORF71 with His-tag in N-terminal position

• Inject purified protein into rabbits for polyclonal antibody production

• Purify antibodies using protein A affinity chromatography

• Validate antibody specificity through:

  • Western blotting against recombinant protein and infected tissue lysates

  • Immunoprecipitation assays

  • Immunofluorescence microscopy to confirm localization patterns

• Test functional blocking capacity in in vitro infection assays

What techniques can determine if ORF71 is involved in viral attachment or entry?

Based on approaches used for other OsHV-1 envelope proteins:

• Produce polyclonal antibodies against ORF71 recombinant protein

• Conduct in vitro neutralization assays:

  • Pre-incubate viral suspension with anti-ORF71 antibodies

  • Expose hemolymph or oyster cells to treated virus

  • Measure viral DNA and RNA detection at 0h, 6h, and 18h post-incubation

  • Compare with untreated virus and other antibody controls

• Perform in vivo inhibition assays:

  • Inject spat with viral suspension pre-incubated with anti-ORF71 antibodies

  • Monitor mortality rates compared to control groups

  • Quantify viral DNA in dead oysters

• Use competition assays with purified recombinant ORF71 to determine specificity

How does hemocyte susceptibility to OsHV-1 affect ORF71-related experimental design?

Research shows that hemocytes from oysters with different genetic backgrounds exhibit varying susceptibility to OsHV-1 infection. When designing experiments to study ORF71:

• Source hemolymph from adult oysters with known susceptibility profiles

• Compare viral RNA/DNA detection between hemolymph from high-susceptibility (e.g., families C and E) and low-susceptibility oysters (e.g., families D and F)

• Measure ORF71 transcript levels alongside control ORFs (such as ORF87, which shows differential expression based on host susceptibility)

• Account for time-dependent expression patterns, with peak viral transcript detection typically occurring at 18h post-incubation

How might ORF71 interact with other OsHV-1 proteins in the viral envelope?

To investigate potential interactions:

• Perform co-immunoprecipitation assays with tagged ORF71 and other viral proteins

• Use proximity labeling techniques (BioID, APEX) to identify interaction partners

• Apply FRET or BiFC to visualize protein-protein interactions in infected cells

• Create truncated variants of ORF71 to map interaction domains

Consider that multiple viral envelope proteins likely work in concert for attachment and entry, as evidenced by the partial inhibition seen with individual antibodies against ORFs 25, 41, and 72, with complete inhibition requiring a combination approach .

What role might RNA editing play in ORF71 expression and function?

Recent transcriptomic research has revealed RNA editing events in OsHV-1 transcripts:

• Analyze ORF71 transcripts for A-to-G variations consistent with adenosine deaminase acting on RNA (ADAR) activity

• Map inosine incorporation sites using deep sequencing approaches

• Determine if ORF71 transcripts undergo hyperediting (concentrated in specific regions) or dispersed single-nucleotide editing

• Investigate the functional consequences of RNA editing on ORF71 protein production and activity

• Assess whether editing represents a viral counter-defense mechanism or host antiviral response

How do genetic variations in ORF71 correlate with virulence across OsHV-1 variants?

To establish structure-function relationships:

What in vitro systems best model ORF71 function in OsHV-1 infection?

Develop experimental systems based on established OsHV-1 research approaches:

• Hemolymph-based assays:

  • Collect hemolymph from adult oysters with known susceptibility profiles

  • Incubate with viral suspension under controlled conditions

  • Monitor viral DNA and RNA levels at defined timepoints (0h, 6h, 18h)

  • Add experimental treatments (antibodies, inhibitors) to assess ORF71 function

• Pseudotyped virus systems:

  • Generate recombinant vesicular stomatitis virus (VSV) or lentivirus expressing ORF71 on surface

  • Assess entry into oyster cells compared to control pseudotypes

  • Use for high-throughput screening of entry inhibitors

How can dextran sulfate inhibition assays be adapted to study ORF71's role in viral attachment?

Dextran sulfate has demonstrated antiviral effects against OsHV-1, likely by interfering with virus-host cell interactions. To investigate ORF71's potential role:

• Perform competitive binding assays:

  • Pre-incubate viral particles with varying concentrations of dextran sulfate (10-50 μg/mL)

  • Add anti-ORF71 antibodies to the mix

  • Assess if dextran sulfate and antibodies show additive or competitive inhibition

• Design functional assays:

  • Compare inhibition profiles of dextran sulfate against wild-type virus and ORF71-depleted virus

  • Measure viral transcript levels at 6h and 18h post-incubation

  • Correlate with mortality rates in in vivo challenge experiments

• Investigate structural interactions:

  • Use surface plasmon resonance to measure binding kinetics between purified ORF71 and dextran sulfate

  • Perform mutation analysis to identify dextran sulfate binding sites on ORF71

How might CRISPR/Cas9 genome editing be applied to study ORF71 function?

For advanced functional genomics approaches:

• Design guide RNAs targeting ORF71 in the OsHV-1 genome

• Generate knockout or mutant viruses using CRISPR/Cas9 editing

• Assess the impact on:

  • Viral replication in hemocyte models

  • Virion assembly and structure

  • Infectivity and pathogenesis in experimental challenges

• Complement with rescue experiments using recombinant ORF71

What transcriptomic approaches can reveal the regulatory network involving ORF71?

Based on recent advances in OsHV-1 transcriptomics:

• Apply Nanopore Direct RNA Sequencing (DRS) to:

  • Map transcription start and stop sites for ORF71

  • Identify potential readthrough events and polycistronic transcripts

  • Detect natural antisense transcripts (NATs) that may regulate ORF71 expression

• Perform temporal transcriptomic analysis to:

  • Place ORF71 within early, immediate-early, or late expression categories

  • Identify co-regulated genes that may function in the same pathway

  • Map the regulatory landscape controlling ORF71 expression