Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that employs several unique strategies for gene expression. The shortest transcript of BDV, X/P mRNA, encodes at least three open reading frames (ORFs): upstream ORF (uORF), X, and P in the 5′ to 3′ direction. The X is a negative regulator of viral polymerase activity, while the P phosphoprotein is a necessary cofactor of the polymerase complex, suggesting that the translation of X is controlled rigorously, depending on viral replication. However, the translation mechanism used by the X/P polycistronic mRNA has not been determined in detail. Here we demonstrate that the X/P mRNA autogenously regulates the translation of X via interaction with host factors. Transient transfection of cDNA clones corresponding to the X/P mRNA revealed that the X ORF is translated predominantly by uORF-termination-coupled reinitiation, the efficiency of which is upregulated by expression of P. We found that P may enhance ribosomal reinitiation at the X ORF by inhibition of the interaction of the DEAD-box RNA helicase DDX21 with the 5′ untranslated region of X/P mRNA, via interference with its phosphorylation. Our results not only demonstrate a unique translational control of viral regulatory protein, but also elucidate a previously unknown mechanism of regulation of polycistronic mRNA translation using RNA helicases.

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Borna disease virus (BDV) is. This observation suggested a unique strategy of intracellular trafficking of a viral protein that is essential for the formation of a functional BDV ribonucleoprotein (RNP). However, neither the mechanism nor the consequences of the cytoplasmic retention or nuclear export of BDV X-P complex have been elucidated. In this study, we show that BDV P contains a bona fide nuclear export signal (NES) and can actively shuttle between the nucleus and cytoplasm. A transient transfection analysis of cDNA clones that mimic the BDV bicistronic X/P mRNA revealed that the methionine-rich (MetR) domain of P is responsible for the X-dependent cytoplasmic localization of the protein complex. Mutational and functional analysis revealed that the methionine residues within the MetR domain are critical for the activity of the NES of P. Furthermore, leptomycin B or small interfering RNA for inhibition of CRM1 strongly suggested that a CRM1-dependent pathway mediates nuclear export of P. Fluorescence loss in photobleaching analysis confirmed the nucleocytoplasmic shuttling of P. Moreover, we revealed that the nuclear export of P is not involved in the inhibition of the polymerase activity by X in the BDV minireplicon system. Our results may provide a unique strategy for the nucleocytoplasmic transport of viral RNP, which could be critical for the formation of not only infectious virions in the cytoplasm but also a persistent viral state in the nucleus.Riboviruses, whose RNA biosynthetic processes, replication, and transcription take place in the nucleus of the infected cell, need to control the directional transport of their genomes through nuclear pore complexes. Recent studies have revealed that viral proteins play central roles in the regulation of nucleocytoplasmic trafficking of viral nucleic acids (2, 24). For instance, the nucleocapsid (NP) and nonstructural (NS2/NEP) proteins of influenza A virus are required for nuclear import and export, respectively, of the viral ribonucleoprotein (RNP) complex (16-18). Likewise, the Rev protein of human immunodeficiency virus type 1 mediates the nuclear transport of unspliced or single-spliced viral transcripts (21). The nucleocytoplasmic transport of viral components is an active and energy-dependent process mediated by specific nuclear localization and export signals termed NLS and NES, respectively, which are present within cargo molecules (2, 7). The interaction between NLS or NES with nuclear transport receptors known as importin and exportin, respectively, regulates the direction in which cargo molecules are transported (7, 23). The signal-dependent translocation of cargo molecules most likely contributes to the nucleocytoplasmic transport of viral components in infected cells, but the detailed mechanisms by which viruses control the directional transport of their genome-protein complexes remain poorly understood.Borna disease virus (BDV) is an enveloped virus with a nonsegmented, negative-strand RNA genome that has a gene organization characteristic of m...