scholarly journals Adaptation of Borna Disease Virus to New Host Species Attributed to Altered Regulation of Viral Polymerase Activity

2007 ◽  
Vol 81 (15) ◽  
pp. 7933-7940 ◽  
Author(s):  
Andreas Ackermann ◽  
Peter Staeheli ◽  
Urs Schneider
Keyword(s):  
Amino Acid ◽  
Disease Virus ◽  
Host Species ◽  
Polymerase Activity ◽  
Viral Polymerase ◽  
New Host ◽  
Borna Disease Virus ◽  
Altered Regulation ◽  
Borna Disease ◽  
New Host Species

ABSTRACT Borna disease virus (BDV) can persistently infect the central nervous system of a broad range of mammalian species. Mice are resistant to infections with primary BDV isolates, but certain laboratory strains can be adapted to replicate in mice. We determined the molecular basis of adaptation by studying mutations acquired by a cDNA-derived BDV strain during one brain passage in rats and three passages in mice. The adapted virus propagated efficiently in mouse brains and induced neurological disease. Its genome contained seven point mutations, three of which caused amino acid changes in the L polymerase (L1116R and N1398D) and in the polymerase cofactor P (R66K). Recombinant BDV carrying these mutations either alone or in combination all showed enhanced multiplication speed in Vero cells, indicating improved intrinsic viral polymerase activity rather than adaptation to a mouse-specific factor. Mutations R66K and L1116R, but not N1398D, conferred replication competence of recombinant BDV in mice if introduced individually. Virus propagation in mouse brains was substantially enhanced if both L mutations were present simultaneously, but infection remained mostly nonsymptomatic. Only if all three amino acid substitutions were combined did BDV replicate vigorously and induce early disease in mice. Interestingly, the virulence-enhancing effect of the R66K mutation in P could be attributed to reduced negative regulation of polymerase activity by the viral X protein. Our data demonstrate that BDV replication competence in mice is mediated by the polymerase complex rather than the viral envelope and suggest that altered regulation of viral gene expression can favor adaptation to new host species.

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 X protein of Borna disease virus regulates viral polymerase activity through interaction with the P protein

2004 ◽  
Vol 85 (7) ◽  
pp. 1895-1898 ◽  
Author(s):  
Marion Poenisch ◽  
Gunhild Unterstab ◽  
Thorsten Wolff ◽  
Peter Staeheli ◽  
Urs Schneider
Keyword(s):  
Disease Virus ◽  
Inhibitory Activity ◽  
Viral Proteins ◽  
Polymerase Activity ◽  
X Protein ◽  
Viral Polymerase ◽  
Interaction Domain ◽  
Borna Disease Virus ◽  
P Protein ◽  
Borna Disease

Borna disease virus polymerase activity is negatively regulated by the viral X protein. Using a virus minireplicon system it was found that all X mutants that no longer interacted with the viral P protein failed to exhibit significant inhibitory activity. The action of X could further be neutralized by expression of a P fragment that contained the X interaction domain but lacked all domains known to mediate interaction with other viral proteins. X thus appears to regulate the activity of the Borna disease virus polymerase by targeting the polymerase cofactor P.

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 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 Feline Lentivirus Evolution in Cross-Species Infection Reveals Extensive G-to-A Mutation and Selection on Key Residues in the Viral Polymerase

2006 ◽  
Vol 80 (6) ◽  
pp. 2728-2737 ◽  
Author(s):  
Mary Poss ◽  
Howard A. Ross ◽  
Sally L. Painter ◽  
David C. Holley ◽  
Julie A. Terwee ◽  
...  
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Host Species ◽  
Puma Concolor ◽  
Cytidine Deaminase ◽  
Productive Infection ◽  
Viral Polymerase ◽  
New Host ◽  
Immunodeficiency Virus ◽  
Key Residues ◽  
New Host Species

ABSTRACT Factors that restrict a virus from establishing productive infection in a new host species are important to understand because cross-species transmission events are often associated with emergent viral diseases. To determine the evolutionary pressures on viruses in new host species, we evaluated the molecular evolution of a feline immunodeficiency virus derived from a wild cougar, Puma concolor, during infection of domestic cats. Analyses were based on the coding portion of genome sequences recovered at intervals over 37 weeks of infection of six cats inoculated by either intravenous or oral-nasal routes. All cats inoculated intravenously, but only one inoculated orally-nasally, became persistently viremic. There were notable accumulations of lethal errors and predominance of G-to-A alterations throughout the genome, which were marked in the viral polymerase gene, pol. Viral structural (env and gag) and accessory (vif and orfA) genes evolved neutrally or were under purifying selection. However, sites under positive selection were identified in reverse transcriptase that involved residues in the nucleotide binding pocket or those contacting the RNA-DNA duplex. The findings of extensive G-to-A alterations in this cross-species infection are consistent with the recently described editing of host cytidine deaminase on lentivirus genomes. Additionally, we demonstrate that the primary site of hypermutation is the viral pol gene and the dominant selective force acting on this feline immunodeficiency virus as it replicates in a new host species is on key residues of the virus polymerase.

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 Expression and Characterization of the Borna Disease Virus Polymerase

2000 ◽  
Vol 74 (9) ◽  
pp. 4425-4428 ◽  
Author(s):  
Michelle Portlance Walker ◽  
Ingo Jordan ◽  
Thomas Briese ◽  
Nicole Fischer ◽  
W. Ian Lipkin
Keyword(s):  
Disease Virus ◽  
Genomic Sequence ◽  
Transcription Unit ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Viral Polymerase ◽  
Borna Disease Virus ◽  
Reading Frame ◽  
Borna Disease

ABSTRACT Borna disease virus is the prototype of a new family,Bornaviridae, within the order Mononegavirales, that is characterized by nuclear transcription, splicing, low level replication, and neurotropism. The products of five open reading frames predicted from the genomic sequence have been confirmed; however, expression of the sixth, corresponding to the putative viral polymerase (L), has not been demonstrated. Here, we describe expression and characterization of a 190-kDa protein proposed to represent L. Expression of this protein from the third transcription unit of the viral genome is dependent on a splicing event that fuses a small upstream open reading frame in frame with the larger downstream continuous open reading frame. The protein is detected by serum antibodies from infected rats and is present in the nucleus, where it colocalizes with the phosphoprotein. L is also shown to be phosphorylated by cellular kinases and to interact with the viral phosphoprotein in coimmunoprecipitation studies. These findings are consistent with the identity of the 190-kDa protein as the viral polymerase and provide insights and describe reagents that will be useful for Bornavirus molecular biology and pathobiology.

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