scholarly journals The X Protein of Borna Disease Virus Serves Essential Functions in the Viral Multiplication Cycle

2007 ◽  
Vol 81 (13) ◽  
pp. 7297-7299 ◽  
Author(s):  
Marion Poenisch ◽  
Sandra Wille ◽  
Andreas Ackermann ◽  
Peter Staeheli ◽  
Urs Schneider
Keyword(s):  
Disease Virus ◽  
Gene Cassette ◽  
Vero Cells ◽  
Polymerase Activity ◽  
Virus Multiplication ◽  
Borna Disease Virus ◽  
Multiplication Cycle ◽  
X Gene ◽  
A Site ◽  
Borna Disease

ABSTRACT The X gene of Borna disease virus (BDV) encodes a nonstructural 10-kDa protein that can interact with viral polymerase cofactor P, thus regulating polymerase activity. It remained unknown whether X is essential for virus multiplication. All our attempts to generate mutant BDV with a nonfunctional X gene proved unsuccessful. However, a mutant virus with an inactive X gene was able to replicate in Vero cells if an artificial gene cassette encoding X was inserted at a site near the 5′ end of the viral genome. These results indicate that X performs essential viral functions.

scholarly journals Selective Virus Resistance Conferred by Expression of Borna Disease Virus Nucleocapsid Components

2003 ◽  
Vol 77 (7) ◽  
pp. 4283-4290 ◽  
Author(s):  
Till Geib ◽  
Christian Sauder ◽  
Sascha Venturelli ◽  
Christel Hässler ◽  
Peter Staeheli ◽  
...  
Keyword(s):  
Disease Virus ◽  
Viral Infections ◽  
Acquired Resistance ◽  
Vero Cells ◽  
Polymerase Activity ◽  
Host Cells ◽  
Borna Disease Virus ◽  
Persistently Infected ◽  
Viral Nucleocapsid ◽  
Borna Disease

ABSTRACT Persistent viral infections can render host cells resistant to superinfection with closely related viruses by largely uncharacterized mechanisms. We present evidence for superinfection exclusion in brains of Borna disease virus (BDV)-infected rats and in persistently infected Vero cells, and we suggest that acquired resistance to BDV is due to unbalanced intracellular levels of viral nucleocapsid components. We observed that expression of BDV protein P, N, or X rendered human cells resistant to subsequent challenge with BDV but not with other RNA viruses, indicating that incorrect stoichiometry of nucleocapsid components selectively blocked the polymerase activity of incoming viruses. Vero cells containing high levels of an untranslatable BDV-N transcript remained virus susceptible, demonstrating that viral protein rather than RNA mediated resistance. Transient overexpression of BDV-P in persistently infected Vero cells was also remarkably effective against BDV, indicating that the intracellular balance of viral nucleocapsid components could serve as a target for future therapeutic antiviral strategies.

scholarly journals Second-site mutations in Borna disease virus overexpressing viral accessory protein X

2009 ◽  
Vol 90 (8) ◽  
pp. 1932-1936 ◽  
Author(s):  
Marion Poenisch ◽  
Sandra Wille ◽  
Urs Schneider ◽  
Peter Staeheli
Keyword(s):  
Disease Virus ◽  
Polymerase Activity ◽  
Viral Fitness ◽  
Fine Tuning ◽  
Borna Disease Virus ◽  
Protein X ◽  
Infected Cells ◽  
X Gene ◽  
Viral Accessory Protein ◽  
Borna Disease

The X protein of Borna disease virus (BDV) is an essential factor that regulates viral polymerase activity and inhibits apoptosis of persistently infected cells. We observed that a BDV mutant which carries an additional X gene replicated well in cell culture only after acquiring second-site mutations that selectively reduced expression of the endogenous X gene. In rat brains, the virus acquired additional mutations which inactivated the ectopic X gene or altered the sequence of X. These results demonstrate that BDV readily acquires mutations if strong selection pressure is applied. They further indicate that fine-tuning of X expression determines viral fitness.

The Borna disease virus (BoDV) 2 nucleoprotein is a conspecific protein that enhances BoDV-1 RNA-dependent RNA polymerase activity

2021 ◽  
Author(s):  
Takehiro Kanda ◽  
Masayuki Horie ◽  
Yumiko Komatsu ◽  
Keizo Tomonaga
Keyword(s):  
Disease Virus ◽  
Reverse Genetics ◽  
Rna Virus ◽  
Polymerase Activity ◽  
Wild Type ◽  
Borna Disease Virus ◽  
Rna Polymerase Activity ◽  
Nucleoprotein N ◽  
Transcription And Replication ◽  
Borna Disease

An RNA virus-based episomal vector (REVec) based on Borna disease virus 1 (BoDV-1) is a promising viral vector that achieves stable and long-term gene expression in transduced cells. However, the onerous procedure of reverse genetics used to generate a REVec is one of the challenges that must be overcome to make REVec technologies practical for use. In this study, to resolve the problems posed by reverse genetics, we focused on BoDV-2, a conspecific virus of BoDV-1 in the Mammalian 1 orthobornavirus . We synthesized the BoDV-2 nucleoprotein (N) and phosphoprotein (P) according to the reference sequences and evaluated their effects on the RNA polymerase activity of the BoDV-1 large protein (L) and viral replication. In the minireplicon assay, we found that BoDV-2 N significantly enhanced BoDV-1 polymerase activity and that BoDV-2 P supported further enhancement of this activity by N. A single amino acid substitution assay identified serine at position 30 of BoDV-2 N and alanine at position 24 of BoDV-2 P as critical amino acid residues for the enhancement of BoDV-1 polymerase activity. In reverse genetics, on the other hand, BoDV-2 N alone was sufficient to increase the rescue efficiency of the REVec. We showed that the REVec can be rescued directly from transfected 293T cells by using BoDV-2 N as a helper plasmid without cocultivation with Vero cells and following several weeks of passage. In addition, a chimeric REVec harboring the BoDV-2 N produced much higher levels of transgene mRNA and genomic RNA than the wild-type REVec in transduced cells. Our results contribute to not only improvements to the REVec system but also understanding of the molecular regulation of orthobornavirus polymerase activity. Importance Borna disease virus 1 (BoDV-1), a prototype virus of the species Mammalian 1 orthobornavirus , is a nonsegmented negative-strand RNA virus that persists in the host nucleus. The nucleoprotein (N) of BoDV-1 encapsidates genomic and antigenomic viral RNA, playing important roles in viral transcription and replication. In this study, we demonstrated that the N of BoDV-2, another genotype in the species Mammalian 1 orthobornavirus , can participate in the viral ribonucleoprotein complex of BoDV-1 and enhance the activity of BoDV-1 polymerase (L) in both the BoDV-1 minireplicon assay and reverse genetics system. Chimeric recombinant BoDV-1 expressing BoDV-2 N but not BoDV-1 N showed higher transcription and replication levels, whereas the propagation and infectious particle production of the chimeric virus were comparable to those of wild-type BoDV-1, suggesting that the level of viral replication in the nucleus is not directly involved in the progeny virion production of BoDVs. Our results demonstrate a molecular mechanism of bornaviral polymerase activity, which will contribute to further development of vector systems using orthobornaviruses.

scholarly journals Active Borna Disease Virus Polymerase Complex Requires a Distinct Nucleoprotein-to-Phosphoprotein Ratio but No Viral X Protein

2003 ◽  
Vol 77 (21) ◽  
pp. 11781-11789 ◽  
Author(s):  
Urs Schneider ◽  
Melanie Naegele ◽  
Peter Staeheli ◽  
Martin Schwemmle
Keyword(s):  
Disease Virus ◽  
Vero Cells ◽  
Expression Vectors ◽  
Regulatory Function ◽  
Functional Assay ◽  
X Protein ◽  
Borna Disease Virus ◽  
N Protein ◽  
Polymerase Complex ◽  
Borna Disease

ABSTRACT Analysis of the composition and regulation of the Borna disease virus (BDV) polymerase complex has so far been limited by the lack of a functional assay. To establish such an assay on the basis of an artificial minigenome, we constructed expression vectors encoding either nucleoprotein (N), phosphoprotein (P), X protein, or polymerase (L) of BDV under the control of the chicken β-actin promoter. A Flag-tagged version of L colocalized with virus-encoded N and P in characteristic nuclear dots of BDV-infected cells and increased viral N-protein levels in persistently infected Vero cells. Vector-driven expression of L, N, and P in BSR-T7 cells together with a negative-sense BDV minigenome carrying a chloramphenicol acetyltransferase (CAT) reporter gene resulted in efficient synthesis of CAT protein. Induction of CAT protein synthesis strongly depended on a 10- to 30-fold molar excess of the N-encoding plasmid over the P-encoding plasmid. Cotransfection of even small amounts of plasmid encoding the viral X protein reduced CAT synthesis to background levels. Thus, the N-to-P stoichiometry seems to play a central role in the regulation of the BDV polymerase complex. Our data further suggest a negative regulatory function for the X protein of BDV.

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

2006 ◽  
Vol 80 (3) ◽  
pp. 1121-1129 ◽  
Author(s):  
Hideyuki Yanai ◽  
Takeshi Kobayashi ◽  
Yohei Hayashi ◽  
Yohei Watanabe ◽  
Naohiro Ohtaki ◽  
...  
Keyword(s):  
Disease Virus ◽  
Nuclear Export ◽  
Rna Virus ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Polymerase Activity ◽  
Cdna Clones ◽  
Borna Disease Virus ◽  
Rich Domain ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that replicates and transcribes in the nucleus of infected cells. Recently, we have demonstrated that BDV phosphoprotein (P) can modulate its subcellular localization through binding to the protein X, which is encoded in the overlapping open reading frame (T. Kobayashi et al., J. Virol. 77:8099-8107, 2003). 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.

scholarly journals Functional Characterization of the Major and Minor Phosphorylation Sites of the P Protein of Borna Disease Virus

2007 ◽  
Vol 81 (11) ◽  
pp. 5497-5507 ◽  
Author(s):  
Sonja Schmid ◽  
Daniel Mayer ◽  
Urs Schneider ◽  
Martin Schwemmle
Keyword(s):  
Disease Virus ◽  
Functional Characterization ◽  
Polymerase Activity ◽  
Viral Rna ◽  
Borna Disease Virus ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
P Proteins ◽  
Borna Disease

ABSTRACT The phosphoprotein P of Borna disease virus (BDV) is an essential cofactor of the viral RNA-dependent RNA polymerase. It is preferentially phosphorylated at serine residues 26 and 28 by protein kinase C ε (PKCε) and, to a lesser extent, at serine residues 70 and 86 by casein kinase II (CKII). To determine whether P phosphorylation is required for viral polymerase activity, we generated P mutants lacking either the PKCε or the CKII phosphate acceptor sites by replacing the corresponding serine residues with alanine (A). Alternatively, these sites were replaced by aspartic acid (D) to mimic phosphorylation. Functional characterization of the various mutants in the BDV minireplicon assay revealed that D substitutions at the CKII sites inhibited the polymerase-supporting activity of P, while A substitutions maintained wild-type activity. Likewise, D substitutions at the PKC sites did not impair the cofactor function of BDV-P, whereas A substitutions at these sites led to increased activity. Interestingly, recombinant viruses could be rescued only when P mutants with modified PKCε sites were used but not when both CKII sites were altered. PKCε mutant viruses showed a reduced capacity to spread in cell culture, while viral RNA and protein expression levels in persistently infected cells were almost normal. Further mutational analyses revealed that substitutions at individual CKII sites were, with the exception of a substitution of A for S86, detrimental for viral rescue. These data demonstrate that, in contrast to other viral P proteins, the cofactor activity of BDV-P is negatively regulated by phosphorylation.

scholarly journals Enhanced polymerase activity confers replication competence of Borna disease virus in mice

2007 ◽  
Vol 88 (11) ◽  
pp. 3130-3132 ◽  
Author(s):  
Andreas Ackermann ◽  
Daniela Kugel ◽  
Urs Schneider ◽  
Peter Staeheli
Keyword(s):  
Enzymatic Activity ◽  
Host Range ◽  
Disease Virus ◽  
Reverse Genetics ◽  
Polymerase Activity ◽  
Viral Glycoprotein ◽  
Viral Polymerase ◽  
Borna Disease Virus ◽  
Polymerase Complex ◽  
Borna Disease

We previously showed that mouse adaptation of cDNA-derived Borna disease virus (BDV) strain He/80FR was associated exclusively with mutations in the viral polymerase complex. Interestingly, independent mouse adaptation of non-recombinant He/80 was correlated with different alterations in the polymerase and mutations in the viral glycoprotein. We used reverse genetics to demonstrate that changes in the polymerase which improve enzymatic activity represent the decisive host range mutations. The glycoprotein mutations did not confer replication competence in mice, although they slightly improved viral performance if combined with polymerase mutations. Our findings suggest that the viral polymerase restricts the host range of BDV.

scholarly journals The negative regulator of Borna disease virus polymerase is a non-structural protein

2005 ◽  
Vol 86 (11) ◽  
pp. 3163-3169 ◽  
Author(s):  
Malte Schwardt ◽  
Daniel Mayer ◽  
Ronald Frank ◽  
Urs Schneider ◽  
Markus Eickmann ◽  
...  
Keyword(s):  
Disease Virus ◽  
Polymerase Activity ◽  
Negative Regulator ◽  
Structural Protein ◽  
Borna Disease Virus ◽  
Virus Particles ◽  
Protein Levels ◽  
Infected Cells ◽  
Nucleoprotein N ◽  
Borna Disease

The X protein of Borna disease virus (BDV) negatively regulates viral polymerase activity. With a BDV mini-replicon system, 30 % inhibition of polymerase activity was observed at an X to phosphoprotein (P) plasmid ratio of 1 : 6 and 100 % inhibition at a ratio of 1 : 1. It was therefore hypothesized that (i) the X : P ratio in infected cells is not significantly higher than 1 : 6 to prevent complete inhibition of polymerase activity and (ii) X is not efficiently incorporated into viral particles, allowing efficient replication early in infection. To test these assumptions, a monoclonal antibody directed against BDV X was generated. Immunofluorescence analysis revealed co-localization of X with the nucleoprotein (N) and P in the nucleus, as well as in the cytoplasm of BDV-infected cells. Quantification of viral protein levels by Western blot analysis, using purified Escherichia coli-derived X, P and N as protein standards, revealed an X : P : N ratio in BDV-infected cells of approximately 1 : 6 : 40. However, only traces of X could be detected in purified BDV stock, suggesting that X is excluded from virus particles. These results indicate that X is a non-structural protein. The lack of X in virus particles may facilitate polymerase activity early in infection; however, the presence of X in persistently infected cells may result in partial inhibition of the polymerase and thus contribute to viral persistence.

scholarly journals A Borna Disease Virus Vector for Expression of Foreign Genes in Neurons of Rodents

2007 ◽  
Vol 81 (13) ◽  
pp. 7293-7296 ◽  
Author(s):  
Urs Schneider ◽  
Andreas Ackermann ◽  
Peter Staeheli
Keyword(s):  
Disease Virus ◽  
Fluorescent Protein ◽  
Foreign Gene ◽  
Borna Disease Virus ◽  
Highly Active ◽  
The Central Nervous System ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
A Site ◽  
Borna Disease

ABSTRACT An expression cassette for green fluorescent protein was successfully inserted at a site near the 5′ end of the genome of Borna disease virus (BDV). When introduced into a mutant virus with highly active polymerase, the foreign gene was strongly expressed in neurons of infected rats. Since BDV can establish long-term persistence in the central nervous system of rodents, it may be used to engineer efficient vectors for specific delivery of foreign genes into highly differentiated neurons.

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