Viral double-stranded RNAs (dsRNAs) responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, profoundly influence a range of host functions in addition to virulence. The 5'-proximal open reading frame, A, of the prototypical hypovirulence-associated viral dsRNA, L-dsRNA, present in hypovirulent strain EP713, was recently shown by DNA-mediated transformation analysis to suppress fungal sporulation, pigmentation, and accumulation of the enzyme laccase (G. H. Choi and D. L. Nuss, EMBO J. 11:473-477, 1992). We mapped this suppressive activity to the autocatalytic papain-like protease, p29, present within the amino-terminal portion of open reading frame A-encoded polyprotein p69. Mutational analysis revealed that the ability of p29 to alter fungal phenotype is dependent upon release from the polyprotein precursor but is independent of intrinsic proteolytic activity. Deletion of the p29-coding domain within the context of an infectious L-dsRNA cDNA clone resulted in a replication-competent viral dsRNA that exhibited intermediate suppressive activity while retaining the ability to confer hypovirulence. Thus, p29 is necessary but not sufficient for the level of virus-mediated suppression of fungal pigmentation, sporulation, and laccase accumulation observed for wild-type hypovirulent strain EP713 and is nonessential for viral RNA replication and virulence attenuation. These results also illustrate the feasibility of engineering infectious viral cDNA for construction of hypovirulent fungal strains with specific phenotypic traits.