The p25 protein encoded by Beet necrotic yellow vein virus (BNYVV) RNA-3 is a pathogenicity determinant that has been implicated in symptom exacerbation on Chenopodiaceae hosts. Several p25 variants exist within natural isolates and p25 sequence variation may influence the degree of pathogenicity of such BNYVV isolates. Expression of p25 from natural A- and P-type isolates in the background of B-type BNYVV cDNA clones gave symptom discrepancies when compared to B-type p25 expression. Such pathogenicity fluctuation was not due to a different subcellular localization of p25 but was correlated with the nature of the tetrad motif present between amino acid residues 67-70, as well as with the capacity of p25 to self-associate and to activate transcription in a yeast one-hybrid system. Our data suggest that the complete sequence of p25 is required for its functions and the identified sequence variations may contribute to correct folding of the protein.