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 Characterization of the P Protein-Binding Domain on the 10-Kilodalton Protein of Borna Disease Virus

2000 ◽  
Vol 74 (7) ◽  
pp. 3413-3417 ◽  
Author(s):  
Tahir H. Malik ◽  
Masahiko Kishi ◽  
Patrick K. Lai
Keyword(s):  
Disease Virus ◽  
Nuclear Localization ◽  
Cell Nucleus ◽  
Binding Domain ◽  
Borna Disease Virus ◽  
Nuclear Localization Signals ◽  
P Protein ◽  
Infected Cells ◽  
Borna Disease

ABSTRACT The Borna disease virus (BDV) is the prototype member of the Bornaviridae, and it replicates in the cell nucleus. The BDV p24P and p40N proteins carry nuclear localization signals (NLS) and are found in the nuclei of infected cells. The BDV p10 protein does not have an NLS, but it binds with P and/or N and is translocated to the nucleus. Hence, p10 may play a role in the replication of BDV in the cell nucleus. Here, we show that the P-binding domain is located in the N terminus of p10 and that S3 and L16 are important for the interaction.

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 Identification and characterization of a new intron in Borna disease virus

2001 ◽  
Vol 82 (3) ◽  
pp. 641-646 ◽  
Author(s):  
Beatrice Cubitt ◽  
Calvin Ly ◽  
Juan Carlos de la Torre
Keyword(s):  
Disease Virus ◽  
Rna Splicing ◽  
Secretory Pathway ◽  
Borna Disease Virus ◽  
Genome Expression ◽  
Molecular Masses ◽  
Infected Cells ◽  
Identification And Characterization ◽  
Borna Disease

Borna disease virus (BDV) has a non-segmented, negative-strand (NNS) RNA genome. In contrast to all other known NNS RNA animal viruses, BDV replication and transcription occur in the nucleus of infected cells. Moreover, BDV uses RNA splicing for the regulation of its genome expression. Two introns (I and II), both present in two viral primary transcripts of 2·5 and 7·2 kb, have been reported in BDV. Here, evidence is provided of a new BDV intron, intron III, generated by alternative 3′ splice-site choice. Intron III-spliced mRNAs were detected at early times post-infection and found to be present in cells from different types and species. Intron III-spliced mRNAs have coding capability for two new viral proteins with predicted molecular masses of 8·4 and 165 (p165) kDa. p165 is a deleted form of the BDV L polymerase, containing three RGD motifs and a signal peptide signal that could target it into the secretory pathway. These findings underscore the proteomic complexity exhibited by BDV.

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 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 Intracerebral Borna Disease Virus Infection of Bank Voles Leading to Peripheral Spread and Reverse Transcription of Viral RNA

PLoS ONE ◽  
2011 ◽  
Vol 6 (8) ◽  
pp. e23622 ◽  
Author(s):  
Paula Maria Kinnunen ◽  
Hanna Inkeroinen ◽  
Mette Ilander ◽  
Eva Riikka Kallio ◽  
Henna Pauliina Heikkilä ◽  
...  
Keyword(s):  
Virus Infection ◽  
Disease Virus ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Borna Disease Virus ◽  
Bank Voles ◽  
Borna Disease

scholarly journals Characterization of an Unusual Importin α Binding Motif in the Borna Disease Virus p10 Protein That Directs Nuclear Import

2002 ◽  
Vol 277 (14) ◽  
pp. 12151-12157 ◽  
Author(s):  
Thorsten Wolff ◽  
Gunhild Unterstab ◽  
Gudrun Heins ◽  
Juergen A. Richt ◽  
Michael Kann
Keyword(s):  
Disease Virus ◽  
Nuclear Import ◽  
Binding Motif ◽  
Borna Disease Virus ◽  
Importin Α ◽  
Borna Disease

scholarly journals Functional Characterization of the Genomic Promoter of Borna Disease Virus (BDV): Implications of 3′-Terminal Sequence Heterogeneity for BDV Persistence

2005 ◽  
Vol 79 (10) ◽  
pp. 6544-6550 ◽  
Author(s):  
Debralee Rosario ◽  
Mar Perez ◽  
Juan Carlos de la Torre
Keyword(s):  
Disease Virus ◽  
Reverse Genetics ◽  
Functional Characterization ◽  
Borna Disease Virus ◽  
Virus Family ◽  
Sequence Heterogeneity ◽  
Enveloped Virus ◽  
A Genome ◽  
Polymerase I ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV) is an enveloped virus with a genome organization characteristic of Mononegavirales. However, based on its unique features, BDV is considered the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. We have described the establishment of a reverse genetics system for the rescue of BDV RNA analogues, or minigenomes, that is based on the use of polymerase I/polymerase II. Using this BDV minigenome rescue system, we have examined the functional implications of the reported sequence heterogeneity found at the 5′ and 3′ termini of the BDV genome and also defined the minimal BDV genomic promoter within the 3′-terminal 25 nucleotides. Our results suggest that the accumulation of RNA genome species containing truncations of one to three nucleotides at their 3′ termini may contribute to modulate BDV RNA replication and gene expression during long-term persistence.

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