The Borna Disease Virus 2 (BoDV-2) Nucleoprotein Is a Conspecific Protein That Enhances BoDV-1 RNA-Dependent RNA Polymerase Activity.

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 an 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, conversely, 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 to 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.

Modeling Borna Disease Virus In Vitro Spread Reveals the Mode of Antiviral Effect Conferred by an Endogenous Bornavirus-Like Element.

Endogenous retroviruses have demonstrated exaptation during long-term evolution with hosts, e.g., resulting in acquisition of antiviral effect on related extant viral infections. While empirical studies have found that an endogenous bornavirus-like element derived from viral nucleoprotein (itEBLN) in the ground squirrel genome shows antiviral effect on virus replication and de novo infection, the antiviral mechanism, dynamics, and quantitative effect of itEBLN remain unknown. In this study, we experimentally and theoretically investigated the dynamics of how an extant bornavirus, Borna disease virus 1 (BoDV-1), spreads and replicates in uninfected, BoDV-1-infected, and itEBLN-expressing cultured cells. Quantifying antiviral effect based on time course data sets, we found that the antiviral effects of itEBLN are estimated to be 75% and 34% on intercellular virus spread and intracellular virus replication, respectively. This discrepancy between intercellular virus spread and intracellular viral replication suggests that viral processes other than the replication of viral ribonucleoprotein complex (RNP) contributed to the suppression of virus spread in itEBLN-expressing cells. Because itEBLN binds to the BoDV-1 RNP, the suppression of viral RNP trafficking can be an attractive candidate explaining this discrepancy.IMPORTANCE Accumulating evidence suggests that some endogenous viral elements (EVEs), including endogenous retroviruses and endogenous nonretroviral virus elements, have acquired functions in the host as a result of long-term coevolution. Recently, an endogenous bornavirus-like element (itEBLN) found in the ground squirrel genome has been shown to have antiviral activity against exogenous bornavirus infection. In this study, we first quantified bornavirus spread in cultured cells and then calculated the antiviral activity of itEBLN on bornavirus infection. The calculated antiviral activity of itEBLN suggests its suppression of multiple processes in the viral life cycle. To our knowledge, this is the first study quantifying the antiviral activity of EVEs and speculating on a model of how some EVEs have acquired antiviral activity during host-virus arms races.

Neuronal retrograde transport of Borna disease virus occurs in signalling endosomes.

Long-range axonal retrograde transport is a key mechanism for the cellular dissemination of neuroinvasive viruses, such as Borna disease virus (BDV), for which entry and egress sites are usually distant from the nucleus, where viral replication takes place. Although BDV is known to disseminate very efficiently in neurons, both in vivo and in primary cultures, the modalities of its axonal transport are still poorly characterized. In this work, we combined different methodological approaches, such as confocal microscopy and biochemical purification of endosomes, to study BDV retrograde transport. We demonstrate that BDV ribonucleoparticles (composed of the viral genomic RNA, nucleoprotein and phosphoprotein), as well as the matrix protein, are transported towards the nucleus into endocytic carriers. These specialized organelles, called signalling endosomes, are notably used for the retrograde transport of neurotrophins and activated growth factor receptors. Signalling endosomes have a neutral luminal pH and thereby offer protection against degradation during long-range transport. This particularity could allow the viral particles to be delivered intact to the cell body of neurons, avoiding their premature release in the cytoplasm.

Borna Disease Virus Assembles Porous Cage-like Viral Factories in the Nucleus.

Animal-derived RNA viruses frequently generate viral factories in infected cells. However, the details of how RNA viruses build such intracellular structures are poorly understood. In this study, we examined the structure and formation of the viral factories, called viral speckle of transcripts (vSPOTs), that are produced in the nuclei of host cells by Borna disease virus (BDV). Super-resolution microscopic analysis showed that BDV assembled vSPOTs as intranuclear cage-like structures with 59-180-nm pores. The viral nucleoprotein formed the exoskeletons of vSPOTs, whereas the other viral proteins appeared to be mainly localized within these structures. In addition, stochastic optical reconstruction microscopy revealed that filamentous structures resembling viral ribonucleoprotein complexes (RNPs) appeared to protrude from the outer surfaces of the vSPOTs. We also found that vSPOTs disintegrated into RNPs concurrently with the breakdown of the nuclear envelope during mitosis. These observations demonstrated that BDV generates viral replication factories whose shape and formation are regulated, suggesting the mechanism of the integrity of RNA virus persistent infection in the nucleus.

Surface glycoprotein of Borna disease virus mediates virus spread from cell to cell.

Borna disease virus (BDV) is a non-segmented negative-stranded RNA virus that maintains a strictly neurotropic and persistent infection in affected end hosts. The primary target cells for BDV infection are brain cells, e.g. neurons and astrocytes. The exact mechanism of how infection is propagated between these cells and especially the role of the viral glycoprotein (GP) for cell-cell transmission, however, are still incompletely understood. Here, we use different cell culture systems, including rat primary astrocytes and mixed cultures of rat brain cells, to show that BDV primarily spreads through cell-cell contacts. We employ a highly stable and efficient peptidomimetic inhibitor to inhibit the furin-mediated processing of GP and demonstrate that cleaved and fusion-active GP is strictly necessary for the cell-to-cell spread of BDV. Together, our quantitative observations clarify the role of Borna disease virus-glycoprotein for viral dissemination and highlight the regulation of GP expression as a potential mechanism to limit viral spread and maintain persistence. These findings furthermore indicate that targeting host cell proteases might be a promising approach to inhibit viral GP activation and spread of infection.

Manipulation of the N-terminal sequence of the Borna disease virus X protein improves its mitochondrial targeting and neuroprotective potential.

To favor their replication, viruses express proteins that target diverse mammalian cellular pathways. Due to the limited size of many viral genomes, such proteins are endowed with multiple functions, which require targeting to different subcellular compartments. One salient example is the X protein of Borna disease virus, which is expressed both at the mitochondria and in the nucleus. Moreover, we recently demonstrated that mitochondrial X protein is neuroprotective. In this study, we sought to examine the mechanisms whereby the X protein transits between subcellular compartments and to define its localization signals, to enhance its mitochondrial accumulation and thus, potentially, its neuroprotective activity. We transfected plasmids expressing fusion proteins bearing different domains of X fused to enhanced green fluorescent protein (eGFP) and compared their subcellular localization to that of eGFP. We observed that the 5-16 domain of X was responsible for both nuclear export and mitochondrial targeting and identified critical residues for mitochondrial localization. We next took advantage of these findings and constructed mutant X proteins that were targeted only to the mitochondria. Such mutants exhibited enhanced neuroprotective properties in compartmented cultures of neurons grown in microfluidic chambers, thereby confirming the parallel between mitochondrial accumulation of the X protein and its neuroprotective potential.-Ferré C. A., Davezac, N., Thouard, A., Peyrin, J. M., Belenguer, P., Miquel, M.-C., Gonzalez-Dunia, D., Szelechowski, M. Manipulation of the N-terminal sequence of the Borna disease virus X protein improves its mitochondrial targeting and neuroprotective potential.

X-linked RNA-binding motif protein (RBMX) is required for the maintenance of Borna disease virus nuclear viral factories.

Borna disease virus (BDV) is a non-segmented, negative-strand RNA virus that establishes persistent infection in the nucleus. Although BDV forms viral inclusion bodies, termed viral speckles of transcripts (vSPOTs), which are associated with chromatin in the nucleus, the host factors involved in the maintenance of vSPOTs remain largely unknown. In this study, we identified X-linked RNA-binding motif protein (RBMX) as a nuclear factor interacting with BDV nucleoprotein. Interestingly, knockdown of RBMX led to disruption of the formation of vSPOTs and reduced both transcription and replication of BDV. Our results indicate that RBMX is involved in the maintenance of the structure of the virus factory in the nucleus.

GC-MS-Based Metabonomic Profiling Displayed Differing Effects of Borna Disease Virus Natural Strain Hu-H1 and Laboratory Strain V Infection in Rat Cortical Neurons.

Borna disease virus (BDV) persists in the central nervous systems of a wide variety of vertebrates and causes behavioral disorders. Previous studies have revealed that metabolic perturbations are associated with BDV infection. However, the pathophysiological effects of different viral strains remain largely unknown. Rat cortical neurons infected with human strain BDV Hu-H1, laboratory BDV Strain V, and non-infected control (CON) cells were cultured in vitro. At day 12 post-infection, a gas chromatography coupled with mass spectrometry (GC-MS) metabonomic approach was used to differentiate the metabonomic profiles of 35 independent intracellular samples from Hu-H1-infected cells (n = 12), Strain V-infected cells (n = 12), and CON cells (n = 11). Partial least squares discriminant analysis (PLS-DA) was performed to demonstrate discrimination between the three groups. Further statistical testing determined which individual metabolites displayed significant differences between groups. PLS-DA demonstrated that the whole metabolic pattern enabled statistical discrimination between groups. We identified 31 differential metabolites in the Hu-H1 and CON groups (21 decreased and 10 increased in Hu-H1 relative to CON), 35 differential metabolites in the Strain V and CON groups (30 decreased and 5 increased in Strain V relative to CON), and 21 differential metabolites in the Hu-H1 and Strain V groups (8 decreased and 13 increased in Hu-H1 relative to Strain V). Comparative metabonomic profiling revealed divergent perturbations in key energy and amino acid metabolites between natural strain Hu-H1 and laboratory Strain V of BDV. The two BDV strains differentially alter metabolic pathways of rat cortical neurons in vitro. Their systematic classification provides a valuable template for improved BDV strain definition in future studies.

Glutamate and lipid metabolic perturbation in the hippocampi of asymptomatic borna disease virus-infected horses.

Borna disease virus (BDV) is a neurotropic, enveloped, non-segmented, negative-stranded RNA virus that infects a wide variety of vertebrate species from birds to humans across a broad global geographic distribution. Animal symptomatology range from asymptomatic infection to behavioral abnormalities to acute meningoencephalitis. Asymptomatic BDV infection has been shown to be more frequent than conventionally estimated. However, the molecular mechanism(s) underyling asymptomatic BDV infection remain largely unknown. Here, based on real-time quantitative PCR and Western blotting, a total of 18 horse hippocampi were divided into BDV-infected (n = 8) and non-infected control (n = 10) groups. A gas chromatography coupled with mass spectrometry (GC-MS) metabolomic approach, in conjunction with multivariate statistical analysis, was used to characterize the hippocampal metabolic changes associated with asymptomatic BDV infection. Multivariate statistical analysis showed a significant discrimination between the BDV-infected and control groups. BDV-infected hippocampi were characterized by lower levels of D-myo-inositol-1-phosphate, glutamate, phosphoethanolamine, heptadecanoic acid, and linoleic acid in combination with a higher level of ammonia. These differential metabolites are primarily involved in glutamate and lipid metabolism. These finding provide an improved understanding of hippocampal changes associated with asymptomatic BDV infection.

Human but Not Laboratory Borna Disease Virus Inhibits Proliferation and Induces Apoptosis in Human Oligodendrocytes In Vitro.

Borna disease virus (BDV) is a neurotropic virus that produces neuropsychiatric dysfunction in a wide range of warm-blooded species. Several studies have associated BDV with human psychiatric illness, but the findings remain controversial. Although oligodendrocytes are a major glial component of brain white matter and play a pivotal role in neuronal cell function, BDV's effects on human oligodendrocytes have not been clarified. Here, the effects of two BDV strains, Hu-H1 (isolated from a bipolar patient) and Strain V (a laboratory strain), on the proliferation and apoptosis of human oligodendrocytes were investigated. Three experimental cell lines were constructed: Hu-H1-infected oligodendroglioma (Hu-H1) cells, Strain V-infected oligodendroglioma (Strain V) cells, and non-infected oligodendroglioma (control) cells. BDV infection was assayed by BDV nucleoprotein (p40) immunofluorescence, cell proliferation was assayed by Cell Counting Kit-8 (CCK8), and cell cycle phases and apoptosis were assayed by flow cytometry. Expressions of the apoptosis-related proteins Bax and Bcl-2 were measured by Western blotting. p40 expression was confirmed in Hu-H1 and Strain V on and after day three post-infection. Strain V cells showed significantly greater cellular proliferation than Hu-H1 cells on and after day three post-infection. In Hu-H1 cells, Bax and Bcl-2 expression were significantly increased and decreased, respectively, on and after day three post-infection. In contrast, in Strain V cells, Bax and Bcl-2 expression were significantly decreased and increased, respectively, on and after day three post-infection. In conclusion, Hu-H1 inhibits cellular proliferation and promotes apoptosis in human oligodendrocytes via Bax upregulation and Bcl-2 downregulation. In contrast, Strain V promotes cellular proliferation and inhibits apoptosis in human oligodendrocytes via Bax downregulation and Bcl-2 upregulation. The effects of the Hu-H1 strain (isolated from a bipolar patient) are opposite from those of Strain V (a laboratory strain), thereby providing a proof of authenticity for both.

TNF-overexpression in Borna disease virus-infected mouse brains triggers inflammatory reaction and epileptic seizures.

Proinflammatory state of the brain increases the risk for seizure development. Neonatal Borna disease virus (BDV)-infection of mice with neuronal overexpression of tumor necrosis factor-α (TNF) was used to investigate the complex relationship between enhanced cytokine levels, neurotropic virus infection and reaction pattern of brain cells focusing on its role for seizure induction. Viral antigen and glial markers were visualized by immunohistochemistry. Different levels of TNF in the CNS were provided by the use of heterozygous and homozygous TNF overexpressing mice. Transgenic TNF, total TNF (native and transgenic), TNF-receptor (TNFR1, TNFR2), IL-1 and N-methyl-D-aspartate (NMDA)-receptor subunit 2B (NR2B) mRNA values were measured by real time RT-PCR. BDV-infection of TNF-transgenic mice resulted in non-purulent meningoencephalitis accompanied by epileptic seizures with a higher frequency in homozygous animals. This correlated with lower weight gain, stronger degree and progression of encephalitis and early, strong microglia activation in the TNF-transgenic mice, most obviously in homozygous animals. Activation of astroglia could be more intense and associated with an unusual hypertrophy in the transgenic mice. BDV-antigen distribution and infectivity in the CNS was comparable in TNF-transgenic and wild-type animals. Transgenic TNF mRNA-expression was restricted to forebrain regions as the transgene construct comprised the promoter of NMDA-receptor subunit2B and induced up-regulation of native TNF mRNA. Total TNF mRNA levels did not increase significantly after BDV-infection in the brain of transgenic mice but TNFR1, TNFR2 and IL-1 mRNA values, mainly in the TNF overexpressing brain areas. NR2B mRNA levels were not influenced by transgene expression or BDV-infection. Neuronal TNF-overexpression combined with BDV-infection leads to cytokine up-regulation, CNS inflammation and glial cell activation and confirmed the presensitizing effect of elevated cytokine levels for the development of spontaneous epileptic seizures when exposed to additional infectious noxi.

Genomic RNAs of Borna disease virus are elongated on internal template motifs after realignment of the 3' termini.

The terminal structures of the Borna disease virus (BDV) genome (vRNA) and antigenome (cRNA) differ from those of other negative strand RNA viruses, as both molecules possess four nucleotides at the 3' terminus without an apparent template at the 5' end of the opposite strand. Consequently, the v- and cRNA molecules are not perfect mirror images, a situation that is not compatible with conventional strategies to maintain genetic information. We show here that recombinant viruses recovered from cDNA lacking the nontemplated nucleotides efficiently reconstitute the 3' overhangs. Analyses of recombinant viruses encoding genetic markers in potential alternative template sequences demonstrated that the BDV v- and cRNA molecules are extended by a realign-and-elongation process on internal template motifs located in close proximity to the 3' ends of v- and cRNA, respectively. The data further suggest that cRNA elongation is restricted to a single template motif of the nascent strand, whereas elongation of vRNA might use multiple template motifs. We propose that the elongation of the 3' termini supports the terminal integrity of the genomic RNA molecules during BDV persistence, and furthermore provides an elegant strategy to eliminate the triphosphate groups from the 5' termini of the BDV v- and cRNA without compromising the genetic information of the virus.

A synthetic cannabinoid agonist promotes oligodendrogliogenesis during viral encephalitis in rats.

Chronic CNS infection by several families of viruses can produce deficits in prefrontal cortex (PFC) and striatal function. Cannabinoid drugs have been long known for their anti-inflammatory properties and their ability to modulate adult neuro and gliogenesis. Therefore, we explored the effects of systemic administration of the cannabinoid agonist WIN55,212-2(WIN) on prefrontal cortex (PFC) and striatal cytogenesis in a viral model of CNS injury and inflammation based on Borna Disease (BD) virus encephalitis. Active BrdU(+) progenitor populations were significantly decreased 1 week after BrdU labeling in BD rats [p<0.001 compared to uninfected (NL) controls] while less than 5% of BrdU(+) cells colabeled for BDV protein. Systemic WIN (1mg/kg i.p. twice daily×7 days) increased the survival of BrdU(+) cells in striatum (p<0.001) and PFC of BD rats, with differential regulation of labeled oligodendroglia precursors vs microglia/macrophages. WIN increased the percentage of BrdU(+) oligodendrocyte precursor cells and decreased BrdU(+) ED-1-labeled phagocytic cells, without producing pro- or antiviral effects. BDV infection decreased the levels of the endocannabinoid anandamide (AEA) in striatum (p<0.05 compared to NL rats), whereas 2-AG levels were unchanged. Our findings indicate that: 1) viral infection is accompanied by alterations of AEA transmission in the striatum, but new cell protection by WIN appears independent of its effect on endocannabinoid levels; and 2) chronic WIN treatment alters the gliogenic cascades associated with CNS injury, promoting oligodendrocyte survival. Limiting reactive gliogenesis and macrophage activity in favor of oliogodendroglia development has significance for demyelinating diseases. Moreover, the ability of cannabinoids to promote the development of biologically supportive or symbiotic oligodendroglia may generalize to other microglia-driven neurodegenerative syndromes including NeuroAIDS and diseases of aging.

Borna disease virus circulating immunocomplex positivity and psychopathology in psychiatric patients in the Czech Republic.

OBJECTIVES: Borna disease virus (BDV) is an RNA virus belonging to the family Bornaviridae. BDV is a neurotropic virus that causes changes in mood, behaviour and cognition. Patients with psychiatric disorders have a higher incidence of BDV positivity than healthy individuals. METHODS: We examined the seropositivity of BDV circulating immunocomplexes (CIC) in psychiatric patients and healthy individuals (blood donors). We examined 39 psychiatric inpatients for the presence of BDV CIC in the serum by ELISA on day 0, 28 and 56. During the same period psychopathology was measured using psychiatric scales (CGI, CGI-I, MADRS, SDS, PANSS). This is the first such study performed in the Czech Republic. RESULTS: BDV CIC positivity was detected in 66.7% of psychiatric patients (26/39) on day 0, in 53.9% (14/26) on day 28 and in 52.9% on day 56 (9/17). The control group was 22.2% (28/126) positive. The incidence of BDV CIC was significantly higher in psychiatric patients than in healthy individuals (p=0.001). The significantly higher level of BDV CIC was associated with the higher severity of psychopathology in comparison with patients with mild or moderate psychopathology (p=0.03). We did not find any association between BDV CIC positivity and other characteristics (age, diagnosis, family, personal history, the history of infectious diseases, contact with animals). CONCLUSION: In our study psychiatric patients had significantly higher levels of BDV CIC than the control group. The highest levels of BDV CIC were detected in patients with more severe psychopathology.

Cell entry of Borna disease virus follows a clathrin-mediated endocytosis pathway that requires Rab5 and microtubules.

Borna disease virus (BDV), the prototypic member of the Bornaviridae family within the order Mononegavirales, exhibits high neurotropism and provides an important and unique experimental model system for studying virus-cell interactions within the central nervous system. BDV surface glycoprotein (G) plays a critical role in virus cell entry via receptor-mediated endocytosis, and therefore, G is a critical determinant of virus tissue and cell tropism. However, the specific cell pathways involved in BDV cell entry have not been determined. Here, we provide evidence that BDV uses a clathrin-mediated, caveola-independent cell entry pathway. We also show that BDV G-mediated fusion takes place at an optimal pH of 6.0 to 6.2, corresponding to an early-endosome compartment. Consistent with this finding, BDV cell entry was Rab5 dependent but Rab7 independent and exhibited rapid fusion kinetics. Our results also uncovered a key role for microtubules in BDV cell entry, whereas the integrity and dynamics of actin cytoskeleton were not required for efficient cell entry of BDV.

Binding properties of GP1 protein of Borna disease virus.

The surface glycoprotein (G) of Borna disease virus (BDV) plays central roles in the process of viral entry. BDV G is cleaved by cellular furin-like proteases into two components, GP1 and GP2. Although GP1 is involved in the virus entry into cells, the binding activity of GP1 to cells is unknown. Therefore, we expressed the wild-type GP1 and a variety of GP1 deletion mutants that were FLAG-tagged at the C-terminus in human embryonic kidney 293T cells. These proteins were then purified using an anti-FLAG antibody and evaluated for their ability to bind to cell lines. GP1 bound to BDV-permissive cells but not to non-permissive cells. GP1 also inhibited BDV infection via its binding to cells. This binding assay should prove useful to map the receptor-binding domain of BDV.

Protein X of Borna disease virus inhibits apoptosis and promotes viral persistence in the central nervous systems of newborn-infected rats.

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-X(A6A7)) failed to block apoptosis in C6 cells. Furthermore, Lewis rats neonatally infected with BDV-X(A6A7) 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-X(A6A7)-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.

Borna disease virus P protein affects neural transmission through interactions with gamma-aminobutyric acid receptor-associated protein.

Borna disease virus (BDV) is one of the infectious agents that causes diseases of the central nervous system in a wide range of vertebrate species and, perhaps, in humans. The phosphoprotein (P) of BDV, an essential cofactor of virus RNA-dependent RNA polymerase, is required for virus replication. In this study, we identified the gamma-aminobutyric acid receptor-associated protein (GABARAP) with functions in neurobiology as one of the viral P protein-interacting cellular factors by using an approach of phage display-based protein-protein interaction analysis. Direct binding between GABARAP and P protein was confirmed by coimmunoprecipitation, protein pull-down, and mammalian two-hybrid analyses. GABARAP originally was identified as a linker between the gamma-aminobutyric acid receptor (GABAR) and the microtubule to regulate receptor trafficking and plays important roles in the regulation of the inhibitory neural transmitter gamma-aminobutyric acid (GABA). We showed that GABARAP colocalizes with P protein in the cells infected with BDV or transfected with the P gene, which resulted in shifting the localization of GABARAP from the cytosol to the nucleus. We further demonstrated that P protein blocks the trafficking of GABAR, a principal GABA-gated ion channel that plays important roles in neural transmission, to the surface of cells infected with BDV or transfected with the P gene. We proposed that during BDV infection, P protein binds to GABARAP, shifts the distribution of GABARAP from the cytoplasm to the nucleus, and disrupts the trafficking of GABARs to the cell membranes, which may result in the inhibition of GABA-induced currents and in the enhancement of hyperactivity and anxiety.

Polymerase read-through at the first transcription termination site contributes to regulation of borna disease virus gene expression.

An unusually long noncoding sequence is located between the N gene of Borna disease virus (BDV) and the genes for regulatory factor X and polymerase cofactor P. Most of these nucleotides are transcribed and seem to control translation of the bicistronic X/P mRNA. We report here that Vero cells persistently infected with mutant viruses containing minor alterations in this control region showed almost normal levels of N, X, and P proteins but exhibited greatly reduced levels of mRNAs coding for these viral gene products. Surprisingly, cells infected with these BDV mutants accumulated a viral transcript 1.9 kb in length that represents a capped and polyadenylated mRNA containing the coding regions of the N, X, and P genes. Cells infected with wild-type BDV also contained substantial amounts of this read-through mRNA, which yielded both N and P protein when translated in vitro. Viruses carrying mutations that promoted read-through transcription at the first gene junction failed to replicate in the brain of adult rats. In the brains of newborn rats, these mutant viruses were able to replicate after acquiring second-site mutations in or near the termination signal located downstream of the N gene. Thus, sequence elements adjacent to the core termination signal seem to regulate the frequency by which the polymerase terminates transcription after the N gene. We conclude from these observations that BDV uses read-through transcription for fine-tuning the expression of the N, X, and P genes which, in turn, influence viral polymerase activity.

Restricted expression of Borna disease virus glycoprotein in brains of experimentally infected Lewis rats.

AIM: Borna disease virus (BDV) induces a persistent infection in the central nervous system (CNS) accompanied by a non-purulent meningoencephalitis. BDV-infection of Lewis rats provides an important model to investigate basic principles of neurotropism, viral persistence and resulting dysfunctions. To date, the in vivo strategies of BDV to persist in the CNS are not fully understood. Viral glycoproteins are main targets of the antiviral defence implicating a controlled expression in case of persistent infections. Therefore, we analysed the expression profiles of the BDV-glycoprotein (BDV-GP) and corresponding BDV-intron II RNA in experimentally infected rat brains, focusing on their spatio-temporal occurrence, regional, cellular and intracellular locations. METHODS: This was carried out by immunohistochemistry and in situ hybridization. The expression pattern of the most abundantly expressed BDV-nucleoprotein (BDV-N) served as a reference. RESULTS: BDV-N mRNA was detected preferentially in the cytoplasm of neurones, whereas BDV-intron II mRNA was found predominantly in the nucleus of brain cells. The genomic RNA was restricted to the nucleus. Expression of BDV-GP was significantly lower than BDV-N expression and mainly limited to cerebral cortex, hippocampus, amygdala and thalamus. BDV-GP was restricted to larger neurones; BDV-N occurred also in astrocytes, oligodendrocytes and ependymal cells. CONCLUSIONS: The expression profiles of BDV-GP, BDV-N and their mRNAs are significantly different, indicating that BDV-GP expression is regulated in vivo. This might be achieved by restricted nuclear export and/or maturation of BDV-intron II mRNA or limited translation as a viral mechanism to escape from the immune response and enable persistence in the CNS.