Potato mop-top virus (PMTV) is the type member of the genus Pomovirus characterized by a tripartite, single-stranded, positive-sense RNA genome. However, PMTV differs from the other pomoviruses by having an open reading frame (ORF) for a 8-kDa cysteine-rich protein (CRP) on RNA 3, downstream the triple gene block. Site-directed mutagenesis of infectious cDNA clones of PMTV showed that the CRP ORF is not needed for the systemic infection in Nicotiana benthamiana and several other hosts. CRP of PMTV was not capable of RNA silencing suppression in agroinfiltration tests. However, the expression of CRP using vectors based on Potato virus X (PVX) or Tobacco mosaic virus (TMV) resulted in necrotic symptoms on N. benthamiana and tobacco plants, respectively. Sequencing of PMTV isolates obtained from the field revealed significant sequence variability of the CRP. These data suggest that the functions of CRP may be expressed only in certain hosts or at certain phases of the PMTV infection cycle.