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Journal of Virology, August 2002, p. 7747-7759, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7747-7759.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Contribution of Protein p40 to Hypovirus-Mediated Modulation of Fungal Host Phenotype and Viral RNA Accumulation

Nobuhiro Suzuki,{dagger} and Donald L. Nuss*

Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, Maryland 20742-4450

Received 27 August 2001/ Accepted 30 April 2002

The papain-like protease p29, derived from the N-terminal portion of the hypovirus CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, was previously shown to contribute to reduced pigmentation and sporulation by the infected host, the chestnut blight fungus Cryphonectria parasitica, while being dispensable for virus replication and attenuation of fungal virulence (hypovirulence). We now report that deletion of the C-terminal portion of p69, which encodes the highly basic protein p40, resulted in replication-competent mutant viruses that were, however, significantly reduced in RNA accumulation. While the {Delta}p40 mutants retained the ability to confer hypovirulence, {Delta}p40-infected fungal strains produced more asexual spores than strains infected with either wild-type CHV1-EP713 or a {Delta}p29 mutant virus. As observed for {Delta}p29-infected colonies, pigment production was significantly increased in {Delta}p40-infected fungal strains relative to that in CHV1-EP713-infected strains. Virus-mediated suppression of laccase production was not affected by p40 deletion. A gain-of-function analysis was employed to map the p40 symptom determinant to the N-terminal domain, encompassing p69 amino acid residues Thr(288) to Arg(312). Evidence that the gain of function was due to the encoded protein rather than the corresponding RNA sequence element was provided by introducing frameshift mutations on either side of the activity determinant domain. Moreover, restoration of symptoms correlated with increased accumulation of viral RNA. These results suggest that p40 indirectly contributes to virus-mediated suppression of fungal pigmentation and conidiation by providing an accessory function in hypovirus RNA amplification. A possible role for p40 in facilitating ORF B expression and the relationship between hypovirus RNA accumulation and symptom expression are discussed.

* Corresponding author. Mailing address: Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, Plant Sciences Building, Room 5115, College Park, MD 20742-4450. Phone: (301) 405-0334. Fax: (301) 314-9075. E-mail: nuss{at}umbi.umd.edu.

{dagger} Present address: Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan.

Journal of Virology, August 2002, p. 7747-7759, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7747-7759.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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