Modulation of Borna Disease Virus Phosphoprotein Nuclear Localization by the Viral Protein X Encoded in the Overlapping Open Reading Frame

Kobayashi, Takeshi; Zhang, Guoqi; Lee, Byeong-Jae; Baba, Satoko; Yamashita, Makiko; Kamitani, Wataru; Yanai, Hideyuki; Tomonaga, Keizō; Ikuta, Kazuyoshi

doi:10.1128/jvi.77.14.8099-8107.2003

Cited by 27 publications

(32 citation statements)

References 48 publications

(100 reference statements)

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“…We have documented that N contains a bona fide NES that utilizes a CRM1-dependent pathway for traveling through the nuclear pore complex (9). We have also shown that the intracellular distribution of BDV P is influenced by its binding to X, which promotes the cytoplasmic accumulation of P (10). Both BDV X and P contain functional NLS, suggesting that the activity of these signals must be impaired in the context of the X-P complex.…”

Section: Discussionmentioning

confidence: 95%

“…Expression vectors encoding X/P-GFP (pgX/P; GFP is green fluorescent protein), P (pgP) and FLAG-tagged P (pcPF) have been previously described (10). Expression vectors pcPHA and pcXHA encoding hemagglutinin (HA)-tagged P and X BDV proteins, respectively, were generated by subcloning the P and X open reading frames, respectively, into the EcoRI and XhoI sites of plasmid pcDNA3 (Invitrogen, San Diego, CA).…”

Section: Methodsmentioning

confidence: 99%

“…We have reported that a P-X interaction might also contribute to the regulation of the nucleocytoplasmic transport of BDV components (10). BDV P contains a bipartite NLS (28), and P can directly bind to all other components of the viral RNP (26,27).…”

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confidence: 99%

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

Yanai

Kobayashi

Hayashi

et al. 2006

J Virol

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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...

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Section: Discussionmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

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

Yanai

Kobayashi

Hayashi

et al. 2006

J Virol

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“…Results from these binding studies indicate that X⅐P⅐L and also X⅐P⅐N complexes are formed. Based on immunofluorescence studies of either BDV-infected cells or transfected cells overexpressing X, it was previously suggested that X might act by sequestration of P to the cytoplasm, thereby limiting the availability of P for viral RNA synthesis in the nucleus (30,32). Although translocation of P into the cytoplasm might partially explain the effect of X, our new data suggest that X could also act more directly in the nucleus, namely by association with the viral polymerase complex through binding to P. This interaction would inhibit the formation of functional polymerase complexes by blocking the association of P⅐L with P⅐N⅐RNA complexes or, alternatively, by blocking the gliding process.…”

Section: Discussionmentioning

confidence: 99%

“…The X interaction domain is located between P residues 72 and 87 (30), and the N interaction domain includes P residues 197-201 (24). Co-transfection of pCA-N (encoding BDV-N) reduced luciferase activity some 5-fold (Fig.…”

Section: Identification Of the L-binding Domain In Bdv-p By Mammalianmentioning

confidence: 99%

Overlap of Interaction Domains Indicates a Central Role of the P Protein in Assembly and Regulation of the Borna Disease Virus Polymerase Complex

Schneider

Blechschmidt

Schwemmle

et al. 2004

Journal of Biological Chemistry

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The active polymerase complex of Borna disease virus is composed of the viral proteins N, P, and L. The viral X (negative regulatory factor) protein acts as a regulator of polymerase activity. Interactions of P with N and X were previously studied, but interactions with L were poorly defined. Using a mammalian two-hybrid system, we observed that L specifically interacts with P but not with N, X, or itself. Mapping of the L-binding domain in the P molecule revealed that it overlaps with two adjacent domains required for multimerization and interaction with N. Competition experiments showed that the interaction between L and P was inefficient when N was present, indicating that L may preferentially interact with free P in infected cells. Interestingly, a multimerization-defective P mutant maintained the ability to interact with L, N, and X but failed to support reporter gene expression from an artificial Borna disease virus minigenome. Furthermore, dominant negative effects on minigenome activity were only observed when P mutants with an intact multimerization domain were used, suggesting that P multimers, rather than monomers, exhibit biological activity. P mutants lacking functional interaction domains for L or N still formed complexes with these viral proteins when wild-type P was available as a bridging molecule, indicating that P multimers have the potential to act as scaffolds on which the RNA polymerase complex is assembled.The active transcription and replication complex of non-segmented negative strand RNA viruses, termed ribonucleoprotein complex (1), consists of the viral polymerase (L), 1 the phosphoprotein (P), the nucleoprotein (N), and the viral RNA (2). P is a co-transcriptional factor of L, whereas N encapsidates the viral genome to form N⅐RNA complexes (3). For Sendai virus, a prototype paramyxovirus, it was shown that P acts as a scaffold protein, which brings L into close proximity to the N⅐RNA complex (4), thus allowing RNA synthesis. In the absence of P, the N⅐RNA complexes are not recognized by L (5). The viral P protein exerts this scaffolding function by employing independent binding sites for L and the N⅐RNA complex (6). P of Sendai virus contains an additional but distinct binding domain for free N (not bound to viral RNA) (7) and a domain required for multimer formation (8, 9). Complex formation between P and free N prevents aggregation of N (7). Oligomerization is an essential prerequisite for Sendai virus P function in viral transcription and replication (4). The precise function of oligomeric P is unclear. It is assumed that oligomer formation facilitates movement of P (and most likely of the complete polymerase complex) on the RNA template, thereby maintaining polymerase processivity (4). A requirement of P oligomers for viral RNA synthesis was also described for P proteins of other viruses of the order Mononegavirales, including vesicular stomatitis virus (10, 11) and human parainfluenza virus type 3 (12). For the P protein of Sendai virus and human parainfluenza virus type 3, a de...

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Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification

Mayer¹,

Baginsky²,

Schwemmle³

2005

Proteomics

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The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.

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Modulation of Borna Disease Virus Phosphoprotein Nuclear Localization by the Viral Protein X Encoded in the Overlapping Open Reading Frame

Cited by 27 publications

References 48 publications

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

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

Overlap of Interaction Domains Indicates a Central Role of the P Protein in Assembly and Regulation of the Borna Disease Virus Polymerase Complex

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification

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