Molecular Ratio between Borna Disease Viral‐p40 and ‐p24 Proteins in Infected Cells Determined by Quantitative Antigen Capture ELISA

Hayashi

et al. 2006

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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...

Section: Discussionmentioning

confidence: 97%

A Methionine-Rich Domain Mediates CRM1-Dependent Nuclear Export Activity of Borna Disease Virus Phosphoprotein

Yanai

Hayashi

et al. 2006

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“…7). In fact, we have recently demonstrated that the molecular ratio between the N and P proteins is significantly different between persistently and acutely BDV-infected cells (48).…”

Section: Discussionmentioning

confidence: 99%

Borna Disease Virus Nucleoprotein Requires both Nuclear Localization and Export Activities for Viral Nucleocytoplasmic Shuttling

Kamitani

Zhang

et al. 2001

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Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV)belongs to the order Mononegavirales and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.Many viruses, including influenza viruses, herpesviruses, and retroviruses, replicate in the nucleus. Therefore, nuclear import and export of the viral genome are critical to the life cycle of viruses in mammalian cells. It has been reported that these virus employ mechanisms by which the viral nucleic acids enter and leave the nucleus in association with their replication stages (51). In human immunodeficiency virus type 1 (HIV-1), viral proteins including integrase, matrix (MA), and Vpr promote localization of the viral preintegration complex (PIC) to the nucleus following the entry of virus into a cell (4,10,33,45). The MA protein harbors nuclear export activity, in addition to nuclear localization activity, to direct the viral RNAs to the cytoplasm (8). Rev proteins of HIV-1 also have both these nuclear transport activities to transport unspliced or single spliced viral transcripts (32). On the other hand, the nucleocapsid protein (NP) and the nonstructural proteins (NS2 and NEP) of influenza A virus are required for nuclear import and export of the viral ribonucleoprotein (RNP) complex, respectively (9, 28-31, 47). Furthermore, matrix protein (M1) of the influenza virus also plays an essential role in RNP nuclear export in combination with other viral proteins (51). Studies of these nuclear transport proteins revealed that they contain specific signals that mediate nuclear transport, called the nuclear localization signal (NLS) and the nuclear export signal (NES), which function independently of the surrounding sequences (27, 51). NLS-or NES-containing proteins can directly or indirectly bind to the viral nucleic acids and travel between the cytoplasm and nucleus through the nuclear pore complex (13...

“…As probes, 6 g of glutathione S-transferase (GST)-p24, GST, or purified p24 protein produced from Escherichia coli per ml was allowed to bind to blotted proteins in PBS-T containing 5% (wt/vol) skimmed milk overnight at 4°C. The expression and purification of the recombinant GST-p24 and removal of GST from GST-p24 were described elsewhere (51). The blots were washed three times with PBS-T for 30 min and were then reacted with anti-GST mouse monoclonal antibody (MAb) or anti-p24 rabbit polyclonal antibody (PAb) in PBS-T containing 5% skimmed milk for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Borna Disease Virus Phosphoprotein Binds a Neurite Outgrowth Factor, Amphoterin/HMG-1

Kamitani

Shoya

et al. 2001

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The Borna disease virus (BDV) p24 phosphoprotein is an abundant protein in BDV-infected cultured cells and animal brains. Therefore, there is a possibility that binding of the p24 protein to cellular factor(s) induces functional alterations of infected neural cells in the brain. To identify a cellular protein(s) that interacts with BDV p24 protein, we performed far-Western blotting with extracts from various cell lines. Using recombinant p24 protein as a probe, we detected a 30-kDa protein in all cell lines examined. Binding between the 30-kDa and BDV p24 proteins was also demonstrated using BDV p24 affinity and ion-exchange chromatography columns. Microsequence analysis of the purified 30-kDa protein revealed that its N terminus showed complete homology with rat amphoterin protein, which is a neurite outgrowth factor abundant in the brain during development. Mammalian two-hybrid and immunoprecipitation analyses also confirmed that amphoterin is a specific target for the p24 protein in vivo. Furthermore, we showed that infection by BDV, as well as purified p24 protein in the medium, significantly decreased cell process outgrowth of cells grown on laminin, indicating the functional inhibition of amphoterin by interaction with the p24 protein. Immunohistochemical analysis revealed decreased levels of amphoterin protein at the leading edges of BDV-infected cells. Moreover, the expression of the receptor for advanced glycation end products, of which the extracellular moiety is a receptor for amphoterin, was not significantly activated in BDV-infected cells during the process of extension, suggesting that the secretion of amphoterin from the cell surface is inhibited by the binding of the p24 protein. These results suggested that BDV infection may cause direct damage in the developing brain by inhibiting the function of amphoterin due to binding by the p24 phosphoprotein.Borna disease virus (BDV) is the prototype of a new family, Bornaviridae, within the nonsegmented negative-strand RNA viruses, the Mononegavirales (12, 45), which is characterized by low productivity, neurotropism, and nuclear localization for transcription and replication (8). Although BDV was originally described as an agent of nonpurulent encephalomyelitis in horses in Germany (40), BDV infection has now been found in a wide range of vertebrate species, including sheep, cattle, cats, and ostriches (6,28,40). Recent epidemiological studies have suggested that BDV infection also occurs in humans and that it may be related to certain psychiatric diseases (7,13,22,27,43). Human BDV was isolated from the peripheral blood granulocyte cell fraction of a psychiatric patient (35). Furthermore, we have also demonstrated BDV infection in the brain of a schizophrenic patient with a very recent onset of disease (32).BDV shows noncytolytic replication in cultured cells. However, neonatal rats infected with BDV develop persistent infection and show developmental disturbances affecting specific areas of the brain (4,9,14,19,42). Neonatal BDV infection also result...