The nucleolus is a prominent subnuclear domain and is classically regarded as the site of transcription of rRNA, processing of the precursor rRNAs and biogenesis of pre-ribosomal particles. In addition to these traditionally recognized activities, the nucleolus also participates in many other aspects of cell function. The umbravirus-encoded ORF3 protein is a multifunctional RNA-binding protein involved in long-distance RNA movement, and protection of viral RNA from RNase attack, including possibly small interfering RNA-guided RNA silencing. In addition to its presence in cytoplasmic ribonucleoprotein particles containing viral RNA, the umbraviral ORF3 protein accumulates in nuclei, preferentially targeting nucleoli. The ORF3 protein domains involved in the localization of the protein to the nucleolus were identified. Functional analysis of the mutants revealed the correlation between the ORF3 protein nucleolar localization and its ability to form the cytoplasmic ribonucleoprotein particles and transport viral RNA long distances via the phloem. Possible mechanisms of the nucleolar involvement in systemic virus infection are discussed.