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.

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 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 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.

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.

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 reverse genetics system for Borna disease virus

2003 ◽  
Vol 84 (11) ◽  
pp. 3099-3104 ◽  
Author(s):  
Mar Perez ◽  
Ana Sanchez ◽  
Beatrice Cubitt ◽  
Debralee Rosario ◽  
Juan Carlos de la Torre
Keyword(s):  
Disease Virus ◽  
Reverse Genetics ◽  
Borna Disease Virus ◽  
Borna Disease

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.

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