The beet necrotic yellow vein virus (BNYVV) RNA-5-encoded p26 protein is involved in the accentuation of symptoms expression of infected Chenopodium quinoa plants and is capable of transcription activation (TA) in yeast. TA was previously localized within the first 55 residues of the p26 protein. Interestingly, TA did not occur when C-terminally deleted forms of p26 were used. We used a genetic screen in the yeast one-hybrid system to select restored TA from randomly generated mutants. The TA domain was found to be located within the first 17 residues. Alanine replacement of aspartic acids 11, 16, and 17 within the full-length p26 prevented TA but did not impair subcellular localization and the symptom expression.