Recombinant Cryphonectria parasitica mycoreovirus 1 Uncharacterized protein VP11 (S11)

What is Mycoreovirus 1 and how does it relate to Cryphonectria parasitica?

Mycoreovirus 1 (MyRV1) is a double-stranded RNA virus belonging to the family Reoviridae that infects Cryphonectria parasitica, the causative agent of chestnut blight disease. MyRV1 has 11 double-stranded RNA genome segments (S1 to S11), each encoding single proteins (VP1 to VP11). This virus confers hypovirulence to C. parasitica, reducing its ability to cause disease in chestnut trees . C. parasitica is a Sordariomycete fungus in the family Cryphonectriaceae that causes devastating disease in American and European chestnut trees .

How is VP11 classified in relation to other MyRV1 proteins?

VP11 is encoded by the smallest genomic segment (S11) of Mycoreovirus 1. While the functions of several other viral proteins have been investigated, VP11 remains largely uncharacterized compared to the proteins encoded by larger segments such as S1, S2, and S3, which are known to undergo intragenic rearrangements . The classification of VP11 as "uncharacterized" indicates that its precise molecular function, structure, and role in viral replication or pathogenicity have not been fully determined using conventional functional analysis methods.

What methodological approaches are typically used to identify uncharacterized viral proteins?

The identification of uncharacterized viral proteins like VP11 typically involves:

• Genomic sequence analysis and open reading frame (ORF) prediction

• Comparison with known protein sequences using BLAST and other homology tools

• Domain and motif searches using programs like InterProScan

• Physicochemical parameter prediction (molecular weight, isoelectric point, etc.)

• Subcellular localization prediction

• Structure prediction using homology modeling

• Functional annotation based on conserved domains

Similar approaches have been successfully applied to annotate uncharacterized proteins in other organisms, where functions were successfully assigned to previously unknown proteins .

What expression systems are most effective for producing recombinant VP11 for functional studies?

For recombinant VP11 production, researchers should consider these methodological approaches:

• Bacterial expression systems (E. coli):

  • Advantages: High yield, simple culture conditions

  • Limitations: May lack proper folding for eukaryotic proteins

  • Recommendation: Use with solubility-enhancing tags (MBP, SUMO)

• Fungal expression systems (especially C. parasitica itself):

  • Advantages: Native cellular environment, proper post-translational modifications

  • Methods: Transformation using hygromycin B or benomyl resistance markers as used in p29 expression studies

• Insect/Baculovirus systems:

  • Advantages: Higher eukaryotic environment with proper folding

  • Recommended for structural studies requiring properly folded protein

The choice depends on downstream applications, with fungal expression being particularly relevant for interaction studies with host factors.

How can genome rearrangements of MyRV1 be detected and analyzed when studying VP11 function?

Genome rearrangements of MyRV1, which may affect S11 and consequently VP11, can be detected and analyzed using these methodological approaches:

• Gel electrophoresis of viral dsRNA:

  • Assess segment size variations through comparison with wild-type electropherotypes

  • Look for newly emerging segments or size shifts in existing segments

• RT-PCR and sequencing:

  • Design primers specific for S11 segment

  • Sequence amplicons to identify internal deletions or duplications

  • Compare with wild-type sequences to characterize rearrangements

• Experimental induction of rearrangements:

  • Co-infection with Cryphonectria hypovirus 1 (CHV1) which contains p29, a protein known to induce rearrangements

  • Expression of p29 in transformants (e.g., Twtp29) to induce rearrangements

  • Monitor effects in RNA silencing-deficient strains (Δdcl2 and Δagl2)

• Analysis workflow:

  • Extract total dsRNA from infected fungal cultures

  • Separate dsRNA segments by electrophoresis

  • Document altered migration patterns

  • Isolate and sequence aberrant segments

  • Compare with wild-type sequence to characterize the rearrangement

These approaches have successfully detected intragenic rearrangements in larger segments (S1-S3) and could be applied to study potential rearrangements in S11 .

What bioinformatic tools can predict the function of uncharacterized proteins like VP11?

Based on methodology used for other uncharacterized proteins, researchers should employ this multi-tool approach:

The efficacy of these databases can be validated using receiver operating characteristics at approximately 83.6% accuracy . This comprehensive approach has successfully annotated previously uncharacterized proteins in other organisms.

How does VP11 potentially interact with other viral proteins in the MyRV1 replication complex?

While specific interactions of VP11 have not been fully characterized in the provided research, a methodological approach to study these interactions should include:

• Yeast two-hybrid screening:

  • Using VP11 as bait against other MyRV1 proteins (VP1-VP10)

  • Screening against a C. parasitica cDNA library to identify host interactions

• Co-immunoprecipitation assays:

  • Expression of tagged VP11 in C. parasitica

  • Pull-down experiments followed by mass spectrometry

• Bimolecular fluorescence complementation:

  • Expression of VP11 fused to half of a fluorescent protein

  • Co-expression with other viral proteins fused to complementary half

  • Visualization of interactions through reconstituted fluorescence

• Analysis of recombination events:

  • Compare frequency of genome rearrangements in S11 versus other segments

  • Determine if p29 from CHV1 affects S11 rearrangements similar to effects observed on S1-S3

These approaches would help elucidate whether VP11 functions independently or as part of the viral replication complex.

How does the host RNA silencing pathway affect expression and function of VP11?

The interaction between VP11 and the host RNA silencing pathway can be studied using these methodological approaches:

• Comparative analysis in wild-type and RNA silencing-deficient strains:

  • Measure VP11 expression levels in wild-type C. parasitica versus Δdcl2 and Δagl2 mutants

  • Assess S11 stability and rearrangement frequency in these genetic backgrounds

• Small RNA profiling:

  • Deep sequencing of small RNAs in infected fungi

  • Mapping small RNAs to the S11 sequence

  • Quantifying siRNA abundance targeting VP11-encoding regions

• CLASH (cross-linking, ligation, and sequencing of hybrids):

  • Identify direct interactions between host Argonaute proteins and viral S11 RNA

Based on research with other MyRV1 segments, we might expect altered expression patterns in RNA silencing-deficient strains, potentially affecting VP11 function and stability .

What is the effect of CHV1 p29 protein on VP11 expression and function?

The multifunctional protein p29 encoded by Cryphonectria hypovirus 1 (CHV1) has been shown to:

• Enhance replication of MyRV1, irrespective of whether it is expressed from the virus genome or host chromosomes

• Induce genome rearrangements in MyRV1 segments

• Function as an RNA silencing suppressor

To study p29's specific effects on VP11:

• Experimental approach:

  • Co-infect C. parasitica with MyRV1 and CHV1

  • Alternatively, express p29 from a transgene in MyRV1-infected fungi

  • Monitor S11 stability and VP11 expression levels

  • Compare with control infections lacking p29

• Expression analysis:

  • Quantitative RT-PCR for S11 RNA levels

  • Western blotting for VP11 protein levels

  • Northern blotting to detect potential S11 rearrangements

Given that p29 has been shown to induce rearrangements in larger segments (S1-S3), researchers should investigate whether similar effects occur with S11, potentially affecting VP11 expression and function .

How can structural biology approaches be used to determine VP11 function?

Advanced structural biology techniques to elucidate VP11 function include:

• X-ray crystallography workflow:

  • Express and purify recombinant VP11 with high purity (>95%)

  • Perform crystallization screening (sparse matrix approach)

  • Optimize crystallization conditions

  • Collect diffraction data and solve structure

  • Analyze structural homology to proteins of known function

• Cryo-electron microscopy:

  • Particularly useful if VP11 forms part of a larger complex

  • Sample preparation with minimal artifacts

  • Single-particle reconstruction

  • Resolution refinement to identify functional domains

• NMR spectroscopy:

  • Suitable for smaller domains of VP11

  • Isotopic labeling of recombinant protein

  • Structure determination in solution state

  • Dynamics studies to identify flexible regions

• Integrative structural biology:

  • Combine multiple techniques with computational modeling

  • Use homology modeling based on similar viral proteins

  • Validate using biochemical and functional assays

These approaches can reveal structural features that suggest function, particularly by identifying catalytic sites or interaction interfaces.

What role might VP11 play in the pathogenicity and host range determination of MyRV1?

The potential role of VP11 in pathogenicity and host range can be investigated through:

• Comparative genomics approach:

  • Analyze S11/VP11 sequences across different MyRV1 isolates

  • Compare with related mycoviruses that infect different fungi

  • Identify conserved regions versus variable regions that might relate to host adaptation

• Experimental host range testing:

  • Generate recombinant MyRV1 with modified S11/VP11

  • Test infection capability in different Cryphonectria species:

    • C. parasitica (primary host)

    • C. naterciae (European species)

    • C. japonica (Asian species)

• Fungal pathogenicity assays:

  • Compare virulence of C. parasitica infected with wild-type versus S11-modified MyRV1

  • Measure lesion formation on Castanea sativa, Quercus robur, and Fagus sylvatica

  • Assess mortality rates and pathogen re-isolation frequencies

• Virulence factor prediction:

  • Computational analysis using tools like VICMPred and VirulentPred

  • Validation through knockout/knockdown experiments

Understanding VP11's contribution to host range would provide insights into cross-species transmission potential of mycoviruses and their application in biological control.

How might CRISPR-Cas9 technologies be applied to study VP11 function?

CRISPR-Cas9 technologies offer several methodological approaches for VP11 research:

• Direct VP11/S11 modification:

  • Engineer an infectious MyRV1 cDNA clone

  • Introduce precise mutations in the S11 segment

  • Create chimeric S11 segments with reporter tags

  • Generate knockout or knockdown systems

• Host factor manipulation:

  • Identify and modify host proteins interacting with VP11

  • Create C. parasitica lines with edited potential VP11 receptors

  • Modify RNA silencing components to study their impact on VP11

• Reverse genetics system for MyRV1:

  • Develop CRISPR-driven recombination for targeted segment replacement

  • Create S11 variants with different functional domains

  • Study effects on viral replication and pathogenicity

• High-throughput screening:

  • CRISPR screening to identify host factors required for VP11 function

  • Create knockout libraries in C. parasitica

  • Select for altered MyRV1 replication phenotypes

These approaches would overcome the limited knowledge of VP11 function and provide direct evidence of its role in viral replication and host interaction.

What emerging techniques could advance our understanding of VP11's role in viral-host interactions?

Emerging research techniques with high potential for VP11 characterization include:

• Single-cell transcriptomics:

  • Analyze heterogeneity in VP11 expression across infected populations

  • Identify correlations between VP11 expression and host response

  • Map temporal dynamics of infection at single-cell resolution

• Proximity labeling proteomics:

  • Express VP11 fused to BioID or APEX2 enzymes

  • Identify proximal proteins in living cells

  • Map the VP11 interaction network with spatial resolution

• Cryo-electron tomography:

  • Visualize VP11 in situ within infected cells

  • Locate VP11 within viral replication factories

  • Determine structural context of VP11 function

• Long-read direct RNA sequencing:

  • Characterize S11 transcripts without amplification bias

  • Identify potential RNA modifications

  • Detect novel splice variants or RNA editing events

• AI-driven protein function prediction:

  • Apply AlphaFold or similar deep learning tools to predict VP11 structure

  • Use machine learning to identify functional motifs from limited data

  • Predict protein-protein or protein-RNA interaction interfaces

These advanced techniques would generate novel insights into VP11 function that traditional approaches might miss, particularly regarding its potential interactions with host defense mechanisms and viral replication complexes.