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.

scholarly journals Viral accessory protein X stimulates the assembly of functional Borna disease virus polymerase complexes

2008 ◽  
Vol 89 (6) ◽  
pp. 1442-1445 ◽  
Author(s):  
Marion Poenisch ◽  
Peter Staeheli ◽  
Urs Schneider
Keyword(s):  
Disease Virus ◽  
Expression Vector ◽  
High Sensitivity ◽  
Viral Proteins ◽  
Accessory Protein ◽  
Viral Mrna ◽  
Borna Disease Virus ◽  
Protein X ◽  
Viral Accessory Protein ◽  
Borna Disease

The Borna disease virus (BDV) proteins X and P are translated from a bicistronic viral mRNA. Here, it was shown that the rescue of recombinant BDV from cDNA was enhanced approximately eightfold if reconstitution of the viral polymerase complex was performed with an expression vector encoding X and P rather than P alone. The results provide evidence that appropriate amounts of X reduce the previously reported high sensitivity of the BDV polymerase to imbalances between the viral proteins N and P. These data indicate that X buffers an unfavourable excess of P, thereby stimulating the assembly of functional BDV polymerase complexes.

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 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 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 Borna Disease Virus Matrix Protein Is an Integral Component of the Viral Ribonucleoprotein Complex That Does Not Interfere with Polymerase Activity

2006 ◽  
Vol 81 (2) ◽  
pp. 743-749 ◽  
Author(s):  
Geoffrey Chase ◽  
Daniel Mayer ◽  
Antonia Hildebrand ◽  
Ronald Frank ◽  
Yohei Hayashi ◽  
...  
Keyword(s):  
Disease Virus ◽  
Matrix Protein ◽  
Polymerase Activity ◽  
Polyclonal Antiserum ◽  
Borna Disease Virus ◽  
Ribonucleoprotein Complex ◽  
Integral Component ◽  
The Matrix ◽  
Infected Cells ◽  
Borna Disease

ABSTRACT We have recently shown that the matrix protein M of Borna disease virus (BDV) copurifies with the affinity-purified nucleoprotein (N) from BDV-infected cells, suggesting that M is an integral component of the viral ribonucleoprotein complex (RNP). However, further studies were hampered by the lack of appropriate tools. Here we generated an M-specific rabbit polyclonal antiserum to investigate the intracellular distribution of M as well as its colocalization with other viral proteins in BDV-infected cells. Immunofluorescence analysis revealed that M is located both in the cytoplasm and in nuclear punctate structures typical for BDV infection. Colocalization studies indicated an association of M with nucleocapsid proteins in these nuclear punctate structures. In situ hybridization analysis revealed that M also colocalizes with the viral genome, implying that M associates directly with viral RNPs. Biochemical studies demonstrated that M binds specifically to the phosphoprotein P but not to N. Binding of M to P involves the N terminus of P and is independent of the ability of P to oligomerize. Surprisingly, despite P-M complex formation, BDV polymerase activity was not inhibited but rather slightly elevated by M, as revealed in a minireplicon assay. Thus, unlike M proteins of other negative-strand RNA viruses, BDV-M seems to be an integral component of the RNPs without interfering with the viral polymerase activity. We propose that this unique feature of BDV-M is a prerequisite for the establishment of BDV persistence.

scholarly journals Detection of a novel Borna disease virus-encoded 10 kDa protein in infected cells and tissues.

1997 ◽  
Vol 78 (10) ◽  
pp. 2459-2466 ◽  
Author(s):  
T Wehner ◽  
H Becht ◽  
K Frese ◽  
J A Richt ◽  
C Herden ◽  
...  
Keyword(s):  
Disease Virus ◽  
Borna Disease Virus ◽  
Infected Cells ◽  
Borna Disease

scholarly journals Authentic Borna disease virus transcripts are spliced less efficiently than cDNA-derived viral RNAs

2000 ◽  
Vol 81 (8) ◽  
pp. 1947-1954 ◽  
Author(s):  
Christian Jehle ◽  
W. Ian Lipkin ◽  
Peter Staeheli ◽  
Rosa M. Marion ◽  
Martin Schwemmle
Keyword(s):  
Disease Virus ◽  
Rna Virus ◽  
Borna Disease Virus ◽  
Viral Rnas ◽  
Negative Strand ◽  
Intron 1 ◽  
Splicing Pattern ◽  
Alternatively Spliced ◽  
Infected Cells ◽  
Borna Disease

Borna disease virus (BDV) is a non-segmented, negative-strand RNA virus that replicates and transcribes its genome in the nucleus of infected cells. It uses the cellular splicing machinery to generate a set of alternatively spliced mRNAs from the 2·8 and 7·1 kb primary transcripts, each harbouring two introns. To determine whether splicing of these transcripts is regulated by viral factors, the extent of splicing was studied in infected cells and COS-7 cells transiently transfected with plasmids encoding the 2·8 kb RNA of BDV. Unspliced RNA was found to be the most abundant RNA species in infected cells, whereas viral transcripts lacking both introns were only found in minute amounts. In sharp contrast, plasmid-derived 2·8 kb RNA was predominantly intron 1-spliced and double-spliced. Co-expression of the BDV proteins P, N and X did not influence splicing of plasmid-expressed 2·8 kb RNA. Furthermore, the splicing pattern did not change when the 2·8 kb RNA was expressed in BDV-infected cells. Based on these results we speculate that splicing of authentic BDV transcripts is tightly linked to transcription by the viral polymerase.

scholarly journals Mechanism of Borna Disease Virus Entry into Cells

1998 ◽  
Vol 72 (1) ◽  
pp. 783-788 ◽  
Author(s):  
Daniel Gonzalez-Dunia ◽  
Beatrice Cubitt ◽  
Juan Carlos de la Torre
Keyword(s):  
Disease Virus ◽  
Virus Entry ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Animal Cells ◽  
Borna Disease Virus ◽  
C Terminus ◽  
Surface Receptor ◽  
Infected Cells ◽  
Borna Disease

ABSTRACT We have investigated the entry pathway of Borna disease virus (BDV). Virus entry was assessed by detecting early viral replication and transcription. Lysosomotropic agents (ammonium chloride, chloroquine, and amantadine), as well as energy depletion, prevented BDV infection, indicating that BDV enters host cells by endocytosis and requires an acidic intracellular compartment to allow membrane fusion and initiate infection. Consistent with this hypothesis, we observed that BDV-infected cells form extensive syncytia upon low-pH treatment. Entry of enveloped viruses into animal cells usually requires the membrane-fusing activity of viral surface glycoproteins (GPs). BDV GP is expressed as two products of 84 and 43 kDa (GP-84 and GP-43, respectively). We show here that only GP-43 is present at the surface of BDV-infected cells and therefore is likely the viral polypeptide responsible for triggering fusion events. We also present evidence that GP-43, which corresponds to the C terminus of GP-84, is generated by cleavage of GP-84 by the cellular protease furin. Hence, we propose that BDV GP-84 is involved in attachment to the cell surface receptor whereas its furin-cleaved product, GP-43, is involved in pH-dependent fusion after internalization of the virion by endocytosis.

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.

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