The virulence properties of potato virus Y (PVY) towards an allelic series at the pvr2 locus in pepper genotypes are related to variations in the genome-linked viral protein (VPg). Eleven amino acid substitutions in the central part of the VPg were identified in strains differing by their virulence properties and were introduced, either singly or in combination, in an infectious PVY clone to get an in-depth genetic analysis of the virulence determinant. The virulence spectrum of these mutants was evaluated by inoculation of four pepper genotypes carrying different alleles at the pvr2 locus. The mutations introduced had complex effects on virulence, including antagonistic epistasis and trade-offs for virulence towards different pvr2 alleles. In addition, several mutants showed new virulence properties that were unknown in the natural environment. Such complex effects of mutations on plant virus virulence are unprecedented. They provide a better understanding of the variable levels of durability of the resistance conferred by the different pvr2 alleles, and have important consequences for a durable management of the resistances.