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

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.

Download Full-text

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

Journal of General Virology ◽

10.1099/vir.0.011841-0 ◽

2009 ◽

Vol 90 (8) ◽

pp. 1932-1936 ◽

Cited By ~ 3

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.

Download Full-text

Modulation of Borna Disease Virus Phosphoprotein Nuclear Localization by the Viral Protein X Encoded in the Overlapping Open Reading Frame

Journal of Virology ◽

10.1128/jvi.77.14.8099-8107.2003 ◽

2003 ◽

Vol 77 (14) ◽

pp. 8099-8107 ◽

Cited By ~ 21

Author(s):

Takeshi Kobayashi ◽

Guoqi Zhang ◽

Byeong-Jae Lee ◽

Satoko Baba ◽

Makiko Yamashita ◽

...

Keyword(s):

Subcellular Localization ◽

Disease Virus ◽

Viral Protein ◽

Rna Virus ◽

Viral Proteins ◽

Borna Disease Virus ◽

Negative Strand ◽

Intracellular Movement ◽

Infected Cells ◽

Borna Disease

ABSTRACT Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that belongs to the Mononegavirales order. Unlike other animal viruses in this order, BDV replicates and transcribes in the nucleus of infected cells. Therefore, regulation of the intracellular movement of virus components must be critical for accomplishing the BDV life cycle in mammalian cells. Previous studies have demonstrated that BDV proteins are prone to accumulate in the nucleus of cells transiently transfected with each expression plasmid of the viral proteins. In BDV infection, however, cytoplasmic distribution of the viral proteins is frequently found in cultured cells and animal brains. In this study, to understand the modulation of subcellular localization of BDV proteins, we investigated the intracellular localization of the viral phosphoprotein (P). Transient-transfection analysis with a cDNA clone corresponding to a bicistronic transcript that expresses both viral X and P revealed that P efficiently localizes in the cytoplasm only when BDV X is expressed in the cells. Furthermore, our analysis revealed that the direct binding between X and P is necessary for the cytoplasmic localization of the P. Interestingly, we showed that X is not detectably expressed in the BDV-infected cells in which P is predominantly found in the nucleus, with little or no signal in the cytoplasm. These observations suggested that BDV P can modulate their subcellular localization through binding to X and that BDV may regulate the expression ratio of each viral product in infected cells to control the intracellular movement of the viral protein complexes. The results presented here provide a new insight into the regulation of the intracellular movement of viral proteins of a unique, nonsegmented, negative-strand RNA virus.

Download Full-text

Splicing-Dependent Subcellular Targeting of Borna Disease Virus Nucleoprotein Isoforms

Journal of Virology ◽

10.1128/jvi.01621-18 ◽

2018 ◽

Vol 93 (5) ◽

Cited By ~ 1

Author(s):

Shohei Kojima ◽

Ryo Sato ◽

Mako Yanai ◽

Yumiko Komatsu ◽

Masayuki Horie ◽

...

Keyword(s):

Disease Virus ◽

Translation Initiation ◽

Rna Viruses ◽

Rna Virus ◽

Viral Proteins ◽

Mrna Splicing ◽

Protein Isoforms ◽

Wild Type ◽

Borna Disease Virus ◽

Borna Disease

ABSTRACTTargeting of viral proteins to specific subcellular compartments is a fundamental step for viruses to achieve successful replication in infected cells. Borna disease virus 1 (BoDV-1), a nonsegmented, negative-strand RNA virus, uniquely replicates and persists in the cell nucleus. Here, it is demonstrated that BoDV nucleoprotein (N) transcripts undergo mRNA splicing to generate truncated isoforms. In combination with alternative usage of translation initiation sites, the N gene potentially expresses at least six different isoforms, which exhibit diverse intracellular localizations, including the nucleoplasm, cytoplasm, and endoplasmic reticulum (ER), as well as intranuclear viral replication sites. Interestingly, the ER-targeting signal peptide in N is exposed by removing the intron by mRNA splicing. Furthermore, the spliced isoforms inhibit viral polymerase activity. Consistently, recombinant BoDVs lacking the N-splicing signals acquire the ability to replicate faster than wild-type virus in cultured cells, suggesting that N isoforms created by mRNA splicing negatively regulate BoDV replication. These results provided not only the mechanism of how mRNA splicing generates viral proteins that have distinct functions but also a novel strategy for replication control of RNA viruses using isoforms with different subcellular localizations.IMPORTANCEBorna disease virus (BoDV) is a highly neurotropic RNA virus that belongs to the orthobornavirus genus. A zoonotic orthobornavirus that is genetically related to BoDV has recently been identified in squirrels, thus increasing the importance of understanding the replication and pathogenesis of orthobornaviruses. BoDV replicates in the nucleus and uses alternative mRNA splicing to express viral proteins. However, it is unknown whether the virus uses splicing to create protein isoforms with different functions. The present study demonstrated that the nucleoprotein transcript undergoes splicing and produces four new isoforms in coordination with alternative usage of translation initiation codons. The spliced isoforms showed a distinct intracellular localization, including in the endoplasmic reticulum, and recombinant viruses lacking the splicing signals replicated more efficiently than the wild type. The results provided not only a new regulation of BoDV replication but also insights into how RNA viruses produce protein isoforms from small genomes.

Download Full-text

Heat shock cognate protein 70 controls Borna disease virus replication via interaction with the viral non-structural protein X

Microbes and Infection ◽

10.1016/j.micinf.2009.01.006 ◽

2009 ◽

Vol 11 (3) ◽

pp. 394-402 ◽

Cited By ~ 19

Author(s):

Yohei Hayashi ◽

Masayuki Horie ◽

Takuji Daito ◽

Tomoyuki Honda ◽

Kazuyoshi Ikuta ◽

...

Keyword(s):

Heat Shock ◽

Disease Virus ◽

Virus Replication ◽

Structural Protein ◽

Borna Disease Virus ◽

Protein X ◽

Heat Shock Cognate Protein ◽

Cognate Protein ◽

Borna Disease

Download Full-text

Nucleocytoplasmic Shuttling of Viral Proteins in Borna Disease Virus Infection

Viruses ◽

10.3390/v5081978 ◽

2013 ◽

Vol 5 (8) ◽

pp. 1978-1990 ◽

Cited By ~ 23

Author(s):

Tomoyuki Honda ◽

Keizo Tomonaga

Keyword(s):

Virus Infection ◽

Disease Virus ◽

Viral Proteins ◽

Nucleocytoplasmic Shuttling ◽

Borna Disease Virus ◽

Borna Disease

Download Full-text

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

Journal of General Virology ◽

10.1099/vir.0.80002-0 ◽

2004 ◽

Vol 85 (7) ◽

pp. 1895-1898 ◽

Cited By ~ 32

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.

Download Full-text

Protein X of Borna Disease Virus Inhibits Apoptosis and Promotes Viral Persistence in the Central Nervous Systems of Newborn-Infected Rats

Journal of Virology ◽

10.1128/jvi.02321-08 ◽

2009 ◽

Vol 83 (9) ◽

pp. 4297-4307 ◽

Cited By ~ 39

Author(s):

Marion Poenisch ◽

Nils Burger ◽

Peter Staeheli ◽

Georg Bauer ◽

Urs Schneider

Keyword(s):

Disease Virus ◽

Cultured Cells ◽

Rna Virus ◽

Viral Persistence ◽

Apoptosis Induction ◽

Wild Type ◽

Borna Disease Virus ◽

Persistently Infected ◽

Protein X ◽

Borna Disease

ABSTRACT Borna disease virus (BDV) is a neurotropic member of the order Mononegavirales with noncytolytic replication and obligatory persistence in cultured cells and animals. Here we show that the accessory protein X of BDV represents the first mitochondrion-localized protein of an RNA virus that inhibits rather than promotes apoptosis induction. Rat C6 astroglioma cells persistently infected with wild-type BDV were significantly more resistant to death receptor-dependent and -independent apoptotic stimuli than uninfected cells or cells infected with a BDV mutant expressing reduced amounts of X. Confocal microscopy demonstrated that X colocalizes with mitochondria and expression of X from plasmid DNA rendered human 293T and mouse L929 cells resistant to apoptosis induction. A recombinant virus encoding a mutant X protein unable to associate with mitochondria (BDV-XA6A7) failed to block apoptosis in C6 cells. Furthermore, Lewis rats neonatally infected with BDV-XA6A7 developed severe neurological symptoms and died around day 30 postinfection, whereas all animals infected with wild-type BDV remained healthy and became persistently infected. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining revealed a significant increase in the number of apoptotic cells in the brain of BDV-XA6A7-infected animals, whereas the numbers of CD3+ T lymphocytes were comparable to those detected in animals infected with wild-type BDV. Our data thus indicate that inhibition of apoptosis by X promotes noncytolytic viral persistence and is required for the survival of cells in the central nervous system of BDV-infected animals.

Download Full-text