N-Terminal Domain of Borna Disease Virus G (p56) Protein Is Sufficient for Virus Receptor Recognition and Cell Entry

Borna disease virus (BDV) surface glycoprotein (GP) (p56) has a predicted molecular mass of 56 kDa. Due to extensive posttranslational glycosylation the protein migrates as a polypeptide of 84 kDa (gp84). The processing of gp84 by the cellular protease furin generates gp43, which corresponds to the C-terminal part of gp84. Both gp84 and gp43 have been implicated in viral entry involving receptor-mediated endocytosis and pH-dependent fusion. We have investigated the domains of BDV p56 involved in virus entry. For this, we used a pseudotype approach based on a recently developed recombinant vesicular stomatitis virus (VSV) in which the gene for green fluorescent protein was substituted for the VSV G protein gene (VSV Delta G*). Complementation of VSV Delta G* with BDV p56 resulted in infectious VSV Delta G* pseudotypes that contained both BDV gp84 and gp43. BDV-VSV chimeric GPs that contained the N-terminal 244 amino acids of BDV p56 and amino acids 421 to 511 of VSV G protein were efficiently incorporated into VSV Delta G* particles, and the resulting pseudotype virions were neutralized by BDV-specific antiserum. These findings indicate that the N-terminal part of BDV p56 is sufficient for receptor recognition and virus entry.     

Age- and host-dependent control of Borna disease virus spread in the developing brains of gerbils and rats

Borna disease virus (BDV) is a non-cytolytic, neurotropic RNA virus that has a broad host range in warm-blooded animals, probably including humans. Recently, we have demonstrated that the neonatal gerbil is a unique model for analyzing BDV-induced acute neurological disease. In this report, to understand the effects of the brain development of gerbils in BDV-induced neuropathogenesis, as well as to investigate the host-dependent differences in BDV propagation and pathogenesis in the brains, we performed experimental infection of BDV using two different infant rodent models, gerbils and rats. We demonstrated here that most of the gerbils infected with BDV on postnatal days (PD) 14, but not on PD1 and PD7, could survive neurological disorders during the observation period of PD85. Interestingly, the levels of BDV RNA and antigen in surviving PD14 inoculated gerbil brains were extremely low, whereas diseased gerbils and both PD7 and PD14 inoculated rats contained significant amounts of BDV antigen in the central nervous system, suggesting that PD14 gerbils successfully controlled BDV spread in the brain. Furthermore, the viral distribution, as well as the expression levels of cytokine and CD8 mRNAs, in the brains was markedly different between the rodent models and between diseased and non-diseased statuses of the gerbils. These results demonstrated that developmentally regulated and host-specific factors could contribute to the prevention of BDV spread in developing animal brains. Studies using different animal systems would provide novel insights into the mechanisms of host defense responses to neurotropic virus infections.     

Borna disease virus glycoprotein is required for viral dissemination in neurons

Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus with a tropism for neurons. Infection with BDV causes neurological diseases in a wide variety of animal species. Although it is known that the virus spreads from neuron to neuron, assembled viral particles have never been visualized in the brains of infected animals. This has led to the hypothesis that BDV spreads as nonenveloped ribonucleoproteins (RNP) rather than as enveloped viral particles. We assessed whether the viral envelope glycoprotein (GP) is required for neuronal dissemination of BDV by using primary cultures of rat hippocampal neurons. We show that upon in vitro infection, BDV replicated and spread efficiently in this system. Despite rapid virus dissemination, very few infectious viral particles were detectable in the culture. However, neutralizing antibodies directed against BDV-GP inhibited BDV spread. In addition, interference with BDV-GP processing by inhibiting furin-mediated cleavage of the glycoprotein blocked virus spread. Finally, antisense treatment with peptide nucleic acids directed against BDV-GP mRNA inhibited BDV dissemination, marking BDV-GP as an attractive target for antiviral therapy against BDV. Together, our results demonstrate that the expression and correct processing of BDV-GP are necessary for BDV dissemination in primary cultures of rat hippocampal neurons, arguing against the hypothesis that the virus spreads from neuron to neuron in the form of nonenveloped RNP.     

Cell-to-cell spread of Borna disease virus proceeds in the absence of the virus primary receptor and furin-mediated processing of the virus surface glycoprotein

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of Mononegavirales. BDV cell entry follows a receptor-mediated endocytosis pathway, which is initiated by the recognition of an as-yet-unidentified receptor at the cell surface by the virus glycoprotein G. BDV G is synthesized as a precursor (GPC) that is cleaved by the cellular protease furin to produce the mature glycoproteins GP1 and GP2, which have been implicated in receptor recognition and pH-dependent fusion events, respectively. BDV is highly neurotropic and its spread in cultured cells proceeds in the absence of detectable extracellular virus or syncytium formation. BDV spread has been proposed to be strictly dependent on the expression and correct processing of BDV G. Here we present evidence that cell-to-cell spread of BDV required neither the expression of cellular receptors involved in virus primary infection, nor the furin-mediated processing of BDV G. We also show that in furin-deficient cells, the release of BDV particles induced by the treatment of BDV-infected cells with hypertonic buffer was not significantly affected, while virion infectivity was dramatically impaired, correlating with the decreased incorporation of BDV G species into viral particles. These findings support the view that the propagation of BDV within the central nervous systems of infected hosts involves both a primary infection that follows a receptor-mediated endocytosis pathway and a subsequent cell-to-cell spread that is independent of the expression of the primary receptor and does not require the processing of BDV G into GP1 and GP2.     

1-beta-D-arabinofuranosylcytosine inhibits borna disease virus replication and spread

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that causes neurological diseases in a variety of warm-blooded animal species. There is general consensus that BDV can also infect humans, being a possible zoonosis. Although the clinical consequences of human BDV infection are still controversial, experimental BDV infection is a well-described model for human neuropsychiatric diseases. To date, there is no effective treatment against BDV. In this paper, we demonstrate that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C), a known inhibitor of DNA polymerases, inhibits BDV replication. Ara-C treatment inhibited BDV RNA and protein synthesis and prevented BDV cell-to-cell spread in vitro. Replication of other negative-strand RNA viruses such as influenza virus or measles virus was not inhibited by Ara-C, underscoring the particularity of the replication machinery of BDV. Strikingly, Ara-C treatment induced nuclear retention of viral ribonucleoparticles. These findings could not be attributed to known effects of Ara-C on the host cell, suggesting that Ara-C directly inhibits the BDV polymerase. Finally, we show that Ara-C inhibits BDV replication in vivo in the brain of infected rats, preventing persistent infection of the central nervous system as well as the development of clinical disease. These findings open the way to the development of effective antiviral therapy against BDV.     

Generation and characterization of a recombinant vesicular stomatitis virus expressing the glycoprotein of Borna disease virus

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. BDV cell entry occurs via receptor-mediated endocytosis, a process initiated by the recognition of an as yet unidentified receptor at the cell surface by the BDV surface glycoprotein (G). The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of cellular receptors and detailed mechanisms involved in BDV cell entry. To overcome this problem, we generated and characterized a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSVDeltaG*/BDVG). Cells infected with rVSVDeltaG*/BDVG produced high titers (10(7) PFU/ml) of cell-free virus progeny, but this virus exhibited a highly attenuated phenotype both in cell culture and in vivo. Attenuation of rVSVDeltaG*/BDVG was associated with a delayed kinetics of viral RNA replication and altered genome/N mRNA ratios compared to results for rVSVDeltaG*/VSVG. Likewise, incorporation of BDV G into virions appeared to be restricted despite its high levels of expression and efficient processing in rVSVDeltaG*/BDVG-infected cells. Notably, rVSVDeltaG*/BDVG recreated the cell tropism and entry pathway of bona fide BDV. Our results indicate that rVSVDeltaG*/BDVG represents a unique tool for the investigation of BDV G-mediated cell entry, as well as the roles of BDV G in host immune responses and pathogenesis associated with BDV infection.     

Borna disease virus persistence causes inhibition of glutamate uptake by feline primary cortical astrocytes

Borna disease virus (BDV), a nonsegmented, negative-stranded (NNS) RNA virus, causes central nervous system (CNS) disease in a broad range of vertebrate species, including felines. Both viral and host factors contribute to very diverse clinical and pathological manifestations associated with BDV infection. BDV persistence in the CNS can cause neurobehavioral and neurodevelopmental abnormalities in the absence of encephalitis. These BDV-induced CNS disturbances are associated with altered cytokine and neurotrophin expression, as well as cell damage that is very restricted to specific brain regions and neuronal subpopulations. BDV also targets astrocytes, resulting in the development of prominent astrocytosis. Astrocytes play essential roles in maintaining CNS homeostasis, and disruption of their normal activities can contribute to altered brain function. Therefore, we have examined the effect of BDV infection on the astrocyte's physiology. We present here evidence that BDV can establish a nonlytic chronic infection in primary cortical feline astrocytes that is associated with a severe impairment in the astrocytes' ability to uptake glutamate. In contrast, the astrocytes' ability to uptake glucose, as well as their protein synthesis, viability, and rate of proliferation, was not affected by BDV infection. These findings suggest that, in vivo, BDV could also affect an important astrocyte function required to prevent neuronal excitotoxicity. This, in turn, might contribute to the neuropathogenesis of BDV.     

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.     

Mechanism of the antiviral action of 1-beta-D-arabinofuranosylcytosine on Borna disease virus

Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus that causes neurological diseases in a variety of warm-blooded animal species. Recently, we showed that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C) was a potent inhibitor of BDV. This finding was surprising for an RNA virus, since Ara-C is a DNA polymerase inhibitor. Thus, we sought to better define the mechanism of action of Ara-C on BDV. Here, we show that (i) this effect is specific for an arabinoside ring carrying a cytosine base, (ii) it requires phosphorylation of the nucleotide, and (iii) it can be reversed by an excess of cytidine. Using the recently described minigenome assay for BDV, we provide evidence suggesting that Ara-C may act as a competitive inhibitor of the BDV replication complex.     

Borna disease virus: a unique pathogen and its interaction with intracellular signalling pathways

Borna disease virus (BDV) is a neurotropic RNA virus that establishes non-cytolytic persistent infection in the central nervous system of warm-blooded animals. Depending on the host species and the route of infection, BDV persistence can modulate neuronal plasticity and animal behaviour and/or may provoke a T cell-mediated immunopathological reaction with high mortality. Therefore, BDV functions as a model pathogen to study persistent virus infection in the central nervous system. Here, we review recent evidence showing that BDV interferes with a spectrum of intracellular signalling pathways, which may be involved in viral spread, maintenance of persistence and modulation of neurotransmitter pathways.     

Maturation of Borna disease virus glycoprotein

The maturation of Borna disease virus (BDV) glycoprotein GP was studied in regard to intracellular compartmentalization, compartmentalization signal-domains, proteolytic processing, and packaging into virus particles. Our data show that BDV-GP is (i) predominantly located in the endoplasmic reticulum (ER), (ii) partially exists in the ER already as cleaved subunits GP-N and GP-C, (iii) is directed to the ER/cis-Golgi region by its transmembrane and/or cytoplasmic domains in CD8-BDV-GP hybrid constructs and (iv) is incorporated in the virus particles as authentic BDV glycoprotein exclusively in the cleaved form decorated with N-glycans of the complex type. Downregulation of BDV-glycoproteins on the cell surface, their limited proteolytic processing, and protection of antigenic epitopes on the viral glycoproteins by host-identical N-glycans are different strategies for persistent virus infections.     

Identification of the amino terminal subunit of the glycoprotein of Borna disease virus

The only surface membrane glycoprotein of Borna disease virus (BDV) is synthesized as a polypeptide with a molecular mass of 57 kDa and N-glycosylated to a precursor glycoprotein (GP) of about 94 kDa. It is processed by the cellular protease furin into the C-terminal membrane-anchored subunit GP-C, also known as gp43, and a presumptive N-terminal subunit GP-N, that is highly glycosylated and has a molecular mass of about 51 kDa. However, up to now the latter remained undetected in BDV-infected material. We describe a novel approach to identify glycan masked linear antigenic epitopes. In the present study, GP-N was identified in BDV-infected cells by a combination of lectin precipitation, enzymatic deglycosylation on blot and immunochemistry using an N-terminal specific antiserum. The GP-N has an apparent molecular mass of 45-50 kDa in its glycosylated form and 27 kDa in its deglycosylated form. N-glycan analysis revealed that the precursor GP contains only mannose-rich N-glycans, whereas GP-N and GP-C contain mannose-rich and complex-type N-glycans.     

Development of a novel Borna disease virus reverse genetics system using RNA polymerase II promoter and SV40 nuclear import signal

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that replicates and transcribes in the nucleus of infected cells. Therefore, efficient synthesis of BDV RNA in the nucleus is critical for the development of a reverse genetics system for this virus. Here, we report the development of such a system using the RNA polymerase II (Pol II) promoter. The BDV minigenome cDNA was flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences and inserted downstream of the Pol II promoter. To improve the efficacy of minigenome expression, we estimated the effects of several signal sequences within the minigenome constructs. We found that insertion of the SV40 nuclear import sequence into the Pol II constructs significantly enhances the replication of the minigenome even in cells lacking the SV40 large T antigen. This novel system is theoretically applicable to any mammalian cell line and would be valuable for analyzing host- or cell-type-dependent differences in BDV replication and production. We could demonstrate here the cell-type-dependent inhibitory effect of the viral protein X on BDV polymerase activity. This system may be useful for various research fields not only of BDV but also of other negative-sense RNA viruses.     

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.Borna disease virus (BDV) causes central nervous system disease in a variety of vertebrate species that is frequently manifested by behavioral abnormalities (27, 59). BDV is the causative agent of Borna disease, an often fatal immune-mediated neurological disease naturally occurring mainly in horses and sheep (21, 26, 47). However, current evidence indicates that the natural host range, prevalence, and geographic distribution of BDV are wider than originally thought (25, 31). Experimentally, BDV has a wide host range, and both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (20, 23, 34, 50). Notably, cases of proventricular dilatation disease affecting different species of psittacine birds have recently been linked to infection with avian bornaviruses (24, 29), a finding that expands the natural host range of bornavirus infections associated with clinical manifestations.BDV is an enveloped virus with a nonsegmented negative-strand RNA genome (11, 33, 53, 55) whose gene organization [3′-N-P-p10 (X)-M-G-L-5′] is characteristic of mononegaviruses. However, on the basis of its unique genetic and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales.The BDV surface glycoprotein G plays a key role in receptor recognition and cell entry (20, 46). The G gene directs the synthesis of a precursor, GPC, with a predicted M_r of ca. 56 kDa, but due to its extensive glycosylation, GPC migrates with an M_r of 84 to 94 kDa. GPC is posttranslationally cleaved by the cellular protease furin into GP-1 and GP-2, corresponding to the N-and C-terminal regions, respectively, of G (2, 8, 19, 49). GP-1 has been shown to be sufficient for virus cell entry via receptor-mediated endocytosis (46), whereas GP-2 likely mediates the pH-dependent fusion event between BDV and cell membranes required for a BDV productive infection (19). In vivo, neurons are the initial target of BDV, suggesting a restricted expression pattern of a yet-unidentified virus receptor. Late in infection, BDV is detected in many tissues and organs as a consequence of its centrifugal spread via the axoplasm of peripheral nerve tissues. Receptor-independent mechanisms also contribute to cell-to-cell propagation of BDV (8).The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of the mechanisms involved in BDV cell entry. To overcome this problem, we generated a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSVΔG*/BDVG) (45). Cells infected with rVSVΔG*/BDVG produced high titers (10⁷ PFU/ml) of cell-free virus progeny. Notably, rVSVΔG*/BDVG recreated the cell tropism and entry pathway of bona fide BDV, thus providing a unique tool for the investigation of BDV G-mediated cell entry.Viruses that enter cells via receptor-mediated endocytosis mainly use trafficking pathways mediated by either clathrin or caveola, although alternative entry pathways have been also reported (36). Nevertheless, clathrin-mediated endocytosis (CME) is the route most commonly used by enveloped viruses for cell internalization (35). The initial virus-cell surface receptor interaction results in the activation of different signaling pathways leading to the accumulation of clathrin coated-pits and subsequent formation of endocytotic vesicles (43). Another major endocytotic pathway used by several viruses, including Ebola virus (16) and SV40 (44), uses caveolae for viral internalization into the cell. This endocytotic pathway is strictly dependent on recruitment of lipid rafts to the cell surface, an event mediated by cholesterol. In this regard, we have recently documented the requirements of cholesterol and structural integrity of cell surface lipid rafts for efficient cell entry of BDV (9).In this work, we provide evidence for the first time that BDV cell entry follows a CME-dependent, caveola-independent pathway. Moreover, we show that BDV entry is Rab5 dependent but Rab7 independent and that BDV G-mediated fusion has a rapid kinetics and an optimal pH between 6.0 and 6.2. These findings indicate that BDV G-mediated fusion occurs within the early-endosome compartment. We also provide evidence that microtubules, but not actin dynamics, play a role in BDV cell entry likely by mediating trafficking of BDV-containing endosomes to the subcellular location where viral and endosomal membranes fuse.     

博尔纳病病毒实时荧光定量PCR诊断试剂盒的评价与应用

目的 评价博尔纳病病毒(Borna disease virus,BDV)实时荧光定量PCR(FQ RT-PCR)试剂盒的各项指标,并了解其实际应用效果.方法 使用BDV OL持续感染细胞株、非BDV病毒序列转染的OL细胞、正常的OL细胞,对BDV RT-PCR试剂盒的敏感性、特异性、重复性和稳定性进行评估,同时检测部分临床病人和动物外周血液RNA.结果 试剂盒可以检测出的病毒RNA最低浓度为10~2,相当于1.5个病毒拷贝数.特异性好,无非特异检出.不同批次的试剂盒的检测结果变异系数接近1.加速破坏的试剂盒和正常试剂盒检测结果之间变异系数在2以内.对临床病人检测阳性率为3.6%,对动物检测阳性率为4.4%.结论 试剂盒敏感性、特异性、重复性和稳定性均佳,是BDV基础研究、流行病学调查、临床检测的良好工具.