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Mapping of a hypovirus p29 protease symptom determinant domain with sequence similarity to potyvirus HC-Pro protease
1Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, Maryland 20742, USA.
Abstract:
Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica results in a spectrum of phenotypic changes that can include alterations in colony morphology and significant reductions in pigmentation, asexual sporulation, and virulence (hypovirulence). Deletion of 88% [Phe(25) to Pro(243)] of the virus-encoded papain-like protease, p29, in the context of an infectious cDNA clone of the prototypic hypovirus CHV1-EP713 (recombinant virus Deltap29) partially relieved virus-mediated suppression of pigmentation and sporulation without altering the level of hypovirulence. We now report mapping of the p29 symptom determinant domain to a region extending from Phe(25) through Gln(73) by a gain-of-function analysis following progressive repair of the Deltap29 deletion mutant. This domain was previously shown to share sequence similarity [including conserved cysteine residues Cys(38), Cys(48), Cys(70), and Cys(72)] with the N-terminal portion of the potyvirus-encoded helper component-proteinase (HC-Pro), a multifunctional protein implicated in aphid-mediated transmission, genome amplification, polyprotein processing, long-distance movement, and suppression of posttranscriptional silencing. Substitution of a glycine residue for either Cys(38) or Cys(48) resulted in no qualitative or quantitative changes in virus-mediated symptoms. Unexpectedly, mutation of Cys(70) resulted in a very severe phenotype that included significantly reduced mycelial growth and profoundly altered colony morphology. In contrast, substitution for Cys(72) resulted in a less severe symptom phenotype approaching that observed for Deltap29. The finding that p29-mediated symptom expression is influenced by two cysteine residues that are conserved in the potyvirus-encoded HC-Pro raises the possibility that these related viral-papain-like proteases function in their respective fungal and plant hosts by impacting ancestrally related regulatory pathways.
Insights
Hypovirus infection causes hypovirulence in chestnut blight fungus. Key cysteine residues in the viral protease p29 influence symptom severity, suggesting conserved regulatory pathways in plant and fungal viruses.
Area of Science:
- Plant Pathology
- Virology
- Mycology
Background:
- Hypovirus infection of *Cryphonectria parasitica* causes hypovirulence, altering pigmentation, sporulation, and virulence.
- The viral protease p29 plays a role in these phenotypic changes, with deletions partially relieving symptom suppression.
Purpose of the Study:
- To map the symptom determinant domain of the hypovirus p29 protease.
- To investigate the role of conserved cysteine residues in p29 function and symptom expression.
Main Methods:
- Construction and analysis of a recombinant hypovirus (Deltap29) with a deleted p29 protease.
- Gain-of-function analysis using progressive repair of the Deltap29 mutant.
- Site-directed mutagenesis of conserved cysteine residues (Cys38, Cys48, Cys70, Cys72) within the p29 determinant domain.
Main Results:
- The p29 symptom determinant domain was mapped to residues Phe(25)-Gln(73).
- Mutations at Cys38 and Cys48 did not alter virus-mediated symptoms.
- Mutation of Cys70 resulted in severe hypovirulence symptoms, including reduced growth and altered morphology.
- Mutation of Cys72 led to a less severe phenotype, similar to Deltap29.
Conclusions:
- The hypovirus p29 protease's symptom expression is modulated by specific cysteine residues, particularly Cys70 and Cys72.
- These findings suggest a potential conserved function of viral papain-like proteases in impacting regulatory pathways across different hosts.
- The study highlights the structural and functional relationship between fungal hypovirus proteases and plant virus HC-Pro proteins.