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Hypovirus Papain-Like Protease p29 Functions in trans To Enhance Viral Double-Stranded RNA Accumulation and Vertical Transmission

Nobuhiro Suzuki,¹ Kazuyuki Maruyama,¹ Miho Moriyama,^2, and Donald L. Nuss^2*

Agrivirology Laboratory, Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan,¹ Center for Biosystems Research, University of Maryland Biotechnology Institute, University of Maryland, College Park, Maryland 20742²

Received 29 May 2003/ Accepted 5 August 2003

The prototypic hypovirus CHV1-EP713 attenuates virulence (hypovirulence) and alters several physiological processes of the chestnut blight fungus Cryphonectria parasitica. The papain-like protease, p29, and the highly basic protein, p40, derived, respectively, from the N-terminal and C-terminal portions of the CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, both contribute to reduced pigmentation and sporulation. The p29 coding region was shown to suppress pigmentation and asexual sporulation in the absence of virus infection in transformed C. parasitica, whereas transformants containing the p40-coding domain exhibited a wild-type, untransformed phenotype. Deletion of either p29 or p40 from the viral genome also results in reduced accumulation of viral RNA. We now show that p29, but not p40, functions in trans to enhance genomic RNA accumulation and vertical transmission of p29 deletion mutant viruses. The frequency of virus transmission through conidia was found to decrease with reduced accumulation of viral genomic double-stranded RNA (dsRNA): from almost 100% for wild-type virus to 50% for p29, and 10 to 20% for p69. When expressed from a chromosomally integrated cDNA copy, p29 elevated viral dsRNA accumulation and transmission for p29 mutant virus to the level shown by wild-type virus. Increased viral RNA accumulation levels were also observed for a p69 mutant lacking almost the entire ORF A sequence. Such enhancements were not detected in transgenic fungal colonies expressing p40. Mutation of p29 residues Cys⁷⁰ or Cys⁷², strictly conserved in hypovirus p29 and potyvirus HC-Pro, resulted in the loss of both p29-mediated suppressive activity in virus-free transgenic C. parasitica and in trans enhancement of RNA accumulation and transmission, suggesting a linkage between these functional activities. These results suggest that p29 is an enhancer of viral dsRNA accumulation and vertical virus transmission through asexual spores.

Present address: Kumamoto Prefectural Agricultural Research Center, Koushi-machi, Kikuchi-gun, Kumamoto 861-1113, Japan.

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