The prototypic hypovirus CHV-1/EP713, responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, encodes two leader papain-like proteases, p29 and p48, at the N-terminus of ORF A and ORF B, respectively. Protease p29 has been shown to be dispensable for hypovirus RNA replication, whereas protease p48 is essential for hypovirus RNA replication. The p48 protein can also act in trans to complement a mutant CHV-1/EP713, p48 deficient virus (δp48). The δp48 mutant virus retained replication competence in the apparent absence of p48 following transmission to wild-type C. parasitica and successive subculturing. It was concluded that p48 plays an essential role in the initiation but not the maintenance of virus RNA propagation. Here, I report efforts to identify the mechanisms by which the CHV-1 p48 papain-like protease contributes to virus replication. Mutational analyses of the p48 catalytic and cleavage site residues defined the requirements for p48/ORF B processing in vivo and revealed its functional importance in the context of viral replication and RNA accumulation. In addition, the ability of ORF B to undergo processing through an alternative pathway was identified. Mutation of either of the p48 catalytic residues eliminated the capability of p48 to complement the δp48 mutant virus, suggesting that these residues are essential for in trans rescue (Chapter 2). Extensive deletion/addition analyses identified two independent but redundant p48 functional domains that are essential for virus replication. The first functional domain is between amino acids Thr169-Glu302 and the second functional domain is between amino acids 330-418. The requirements for p48 function were found to be different when p48 was provided in trans than when provided in cis . Whereas only one functional domain is required for viral replication when p48 is supplied in cis, neither functional domain alone is sufficient for complementation of the δp48 mutant virus (Chapter 3). The information gained from these studies has significantly increased our knowledge of the requirements and role of p48 in hypovirus replication and provided new insights into the diversity of replication strategies employed by positive strand RNA viruses.