Borna Disease Virus Replication Research Articles

Suppression of Borna Disease Virus Replication during Its Persistent Infection Using the CRISPR/Cas13b System.

Borna disease virus (BoDV-1) is a bornavirus that infects the central nervous systems of various animal species, including humans, and causes fatal encephalitis. BoDV-1 also establishes persistent infection in neuronal cells and causes neurobehavioral abnormalities. Once neuronal cells or normal neural networks are lost by BoDV-1 infection, it is difficult to regenerate damaged neural networks. Therefore, the development of efficient anti-BoDV-1 treatments is important to improve the outcomes of the infection. Recently, one of the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) systems, CRISPR/Cas13, has been utilized as antiviral tools. However, it is still unrevealed whether the CRISPR/Cas13 system can suppress RNA viruses in persistently infected cells. In this study, we addressed this question using persistently BoDV-1-infected cells. The CRISPR/Cas13 system targeting viral mRNAs efficiently decreased the levels of target viral mRNAs and genomic RNA (gRNA) in persistently infected cells. Furthermore, the CRISPR/Cas13 system targeting viral mRNAs also suppressed BoDV-1 infection if the system was introduced prior to the infection. Collectively, we demonstrated that the CRISPR/Cas13 system can suppress BoDV-1 in both acute and persistent infections. Our findings will open the avenue to treat prolonged infection with RNA viruses using the CRISPR/Cas13 system.

MiR-505 inhibits replication of Borna disease virus 1 via inhibition of HMGB1-mediated autophagy.

Borna disease virus 1 (BoDV-1) is a highly neurotropic RNA virus which was recently demonstrated to cause deadly human encephalitis. Viruses can modulate microRNA expression, in turn modulating cellular immune responses and regulating viral replication. A previous study indicated that BoDV-1 infection down-regulated the expression of miR-505 in rats. However, the underlying mechanism of miR-505 during BoDV-1 infection remains unknown. In this study, we found that miR-505 can inhibit autophagy activation by down-regulating the expression of its target gene HMGB1, and ultimately inhibit the replication of BoDV-1. Specifically, we found that the expression of miR-505 was significantly down-regulated in rat primary neurons stably infected with BoDV-1. Overexpression of miR-505 can inhibit the replication of BoDV-1 in cells. Bioinformatics analysis and dual luciferase reporter gene detection confirmed that during BoDV-1 infection, the high-mobility group protein B1 (HMGB1) that mediates autophagy is the direct target gene of miR-505. The expression of HMGB1 was up-regulated after BoDV-1 infection, and overexpression of miR-505 could inhibit the expression of HMGB1. Autophagy-related detection found that after infection with BoDV-1, the expression of autophagy-related proteins and autophagy-related marker LC3 in neuronal cells was significantly up-regulated. Autophagy flow experiments and transmission electron microscopy also further confirmed that BoDV-1 infection activated HMGB1-mediated autophagy. Further regulating the expression of miR-505 found that overexpression of miR-505 significantly inhibited HMGB1-mediated autophagy. The discovery of this mechanism may provide new ideas and directions for the prevention and treatment of BoDV-1 infection in the future.

Intracellular dynamics of actin affects Borna disease virus replication in the nucleus

Borna disease virus (BoDV) is a nonsegmented, negative-strand RNA virus that uniquely replicates and establishes persistent infection in cell nucleus. Recent studies have demonstrated the presence of actin in the nucleus and its role in intranuclear phenomena such as transcription and DNA repair. Although nuclear actin is involved in the life cycle of some intranuclear DNA viruses, the interaction between BoDV and nuclear actin has not been reported. In this study, we show that the inhibition of the nucleocytoplasmic transport of actin affects the replication of BoDV in the nucleus. The knockdown of a nuclear export factor of actin, exportin 6, results in the induction of structural aberration in intranuclear viral factories of BoDV. Furthermore, the inhibition of the nuclear export of actin promotes accumulation of viral matrix protein in the cytoplasm and periphery of the infected cells. These results suggest that the dynamics of actin affect the replication of BoDV by disturbing the structure of viral factories in the nucleus.

Linkage between the leader sequence and leader RNA production in Borna disease virus-infected cells

The 3′-untranslated region (UTR) of the non-segmented, negative-strand (NNS) RNA viral genome is called the leader sequence, and functions as the promoter for viral replication and transcription. NNS RNA viruses also use the sequence as a template to synthesize leader RNAs (leRNAs) with unknown functions. Borna disease virus (BDV) is unique because it establishes a persistent infection and replicates in the nucleus. No report has yet demonstrated the presence of leRNAs during BDV infection. Here, we report that BDV synthesizes leRNAs from the 3′-UTR of the genome. They started at position 5 in the 3′-UTR and ended by the transcription start signal of the nucleoprotein gene. The level of leRNA production is not correlated with the levels of viral replication and transcription. On the other hand, mutation of the 3′-UTR affects leRNA production. Our findings add a novel viral transcript to the BDV life cycle and shed light on BDV replication and/or transcription.

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.

Heat stress is a potent stimulus for enhancing rescue efficiency of recombinant Borna disease virus.

Recently developed vector systems based on Borna disease virus (BDV) hold promise as platforms for efficient and stable gene delivery to the central nervous system (CNS). However, because it currently takes several weeks to rescue recombinant BDV (rBDV), an improved rescue procedure would enhance the utility of this system. Heat stress reportedly enhances the rescue efficiency of other recombinant viruses. Here, heat stress was demonstrated to increase the amount of BDV genome in persistently BDV-infected cells without obvious cytotoxicity. Further analyses suggested that the effect of heat stress on BDV infection is not caused by an increase in the activity of BDV polymerase. More cells in which BDV replication occurs were obtained in the initial phase of rBDV rescue by using heat stress than when it was not used. Thus, heat stress is a useful improvement on the published rescue procedure for rBDV. The present findings may accelerate the practical use of BDV vector systems in basic science and the clinic and thus enable broader adoption of this viral vector, which is uniquely suited for gene delivery to the CNS.

Inhibition of Borna disease virus replication by an endogenous bornavirus-like element in the ground squirrel genome.

Animal genomes contain endogenous viral sequences, such as endogenous retroviruses and retrotransposons. Recently, we and others discovered that nonretroviral viruses also have been endogenized in many vertebrate genomes. Bornaviruses belong to the Mononegavirales and have left endogenous fragments, called "endogenous bornavirus-like elements" (EBLs), in the genomes of many mammals. The striking features of EBLs are that they contain relatively long ORFs which have high sequence homology to the extant bornavirus proteins. Furthermore, some EBLs derived from bornavirus nucleoprotein (EBLNs) have been shown to be transcribed as mRNA and probably are translated into proteins. These features lead us to speculate that EBLs may function as cellular coopted genes. An EBLN element in the genome of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), itEBLN, encodes an ORF with 77% amino acid sequence identity to the current bornavirus nucleoprotein. In this study, we cloned itEBLN from the ground squirrel genome and investigated its involvement in Borna disease virus (BDV) replication. Interestingly, itEBLN, but not a human EBLN, colocalized with the viral factory in the nucleus and appeared to affect BDV polymerase activity by being incorporated into the viral ribonucleoprotein. Our data show that, as do certain endogenous retroviruses, itEBLN potentially may inhibit infection by related exogenous viruses in vivo.

Absence of a robust innate immune response in rat neurons facilitates persistent infection of Borna disease virus in neuronal tissue.

Borna disease virus (BDV) persistently infects neurons of the central nervous system of various hosts, including rats. Since type I IFN-mediated antiviral response efficiently blocks BDV replication in primary rat embryo fibroblasts, it has been speculated that BDV is not effectively sensed by the host innate immune system in the nervous system. To test this assumption, organotypical rat hippocampal slice cultures were infected with BDV for up to 4 weeks. This resulted in the secretion of IFN and the up-regulation of IFN-stimulated genes. Using the rat Mx protein as a specific marker for IFN-induced gene expression, astrocytes and microglial cells were found to be Mx positive, whereas neurons, the major cell type in which BDV is replicating, lacked detectable levels of Mx protein. In uninfected cultures, neurons also remained Mx negative even after treatment with high concentrations of IFN-α. This non-responsiveness correlated with a lack of detectable nuclear translocation of both pSTAT1 and pSTAT2 in these cells. Consistently, neuronal dissemination of BDV was not prevented by treatment with IFN-α. These data suggest that the poor innate immune response in rat neurons renders this cell type highly susceptible to BDV infection even in the presence of exogenous IFN-α. Intriguingly, in contrast to rat neurons, IFN-α treatment of mouse neurons resulted in the up-regulation of Mx proteins and block of BDV replication, indicating species-specific differences in the type I IFN response of neurons between mice and rats.

Modulation of miR-122 on persistently Borna disease virus infected human oligodendroglial cells

Using RNAhybrid software we found the predicted binding of complementary sequences between miR-122 and viral mRNAs, may be important for the antiviral effect of miR-122 on Borna disease virus (BDV). A moderate expression of miR-122 was identified in human oligodendroglial cells (OL), but with a much lower level of miR-122 in BDV persistent infection (OL/BDV) and cells transfected with BDV gene expression vectors. Over-expression of miR-122 and specific blocking experiments demonstrated that miR-122 was able to specifically inhibit BDV protein synthesis, viral gene replication and transcription, and induce the secretion/synthesis of interferon (IFN) in OL and OL/BDV cells. The abolishment of miR-122 by AMO-122 inhibited endogenous IFN induction by IFN-beta. These results indicate that miR-122 can exert direct antiviral function by inhibiting BDV translation and replication on one hand, while acting indirectly through IFN to increase the host innate immunity to modulate the virus–host interactions on the other hand.

Interferon-γ Prevents Death of Bystander Neurons during CD8 T Cell Responses in the Brain

T cells restricted to neurotropic viruses are potentially harmful as their activity may result in the destruction of neurons. In the Borna disease virus (BDV) model, antiviral CD8 T cells entering the brain of infected mice cause neurological disease but no substantial loss of neurons unless the animals lack interferon-gamma (IFN-gamma). We show here that glutamate receptor antagonists failed to prevent BDV-induced neuronal loss in IFN-gamma-deficient mice, suggesting that excitotoxicity resulting from glutamate receptor overstimulation is an unlikely explanation for the neuronal damage. Experiments with IFN-gamma-deficient mice lacking eosinophils indicated that these cells, which specifically accumulate in the infected brains of IFN-gamma-deficient mice, are not responsible for CA1 neuronal death. Interestingly, BDV-induced damage of CA1 neurons was reduced significantly in IFN-gamma-deficient mice lacking perforin, suggesting a key role for CD8 T cells in this pathological process. Specific death of hippocampal CA1 neurons could be triggered by adoptive transfer of BDV-specific CD8 T cells from IFN-gamma-deficient mice into uninfected mice that express transgene-encoded BDV antigen at high level in astrocytes. These results indicate that attack by CD8 T cells that cause the death of CA1 neurons might be directed toward regional astrocytes and that IFN-gamma protects vulnerable CA1 neurons from collateral damage resulting from exposure to potentially toxic substances generated as a result of CD8 T cell-mediated impairment of astrocyte function.

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

Borna disease virus (BDV) is a non-segmented, negative-sense RNA virus and has the property of persistently infecting the cell nucleus. BDV encodes a 10-kDa non-structural protein, X, which is a negative regulator of viral polymerase activity but is essential for virus propagation. Recently, we have demonstrated that interaction of X with the viral polymerase cofactor, phosphoprotein (P), facilitates translocation of P from the nucleus to the cytoplasm. However, the mechanism by which the intracellular localization of X is controlled remains unclear. In this report, we demonstrate that BDV X interacts with the 71 kDa molecular chaperon protein, Hsc70. Immunoprecipitation assays revealed that Hsc70 associates with the same region of X as P and, interestingly, that expression of P interferes competitively with the interaction between X and Hsc70. A heat shock experiment revealed that BDV X translocates into the nucleus, dependent upon the nuclear accumulation of Hsc70. Furthermore, we show that knockdown of Hsc70 by short interfering RNA decreases the nuclear localization of both X and P and markedly reduces the expression of viral genomic RNA in persistently infected cells. These data indicate that Hsc70 may be involved in viral replication by regulating the intracellular distribution of X.

Ribavirin inhibits Borna disease virus proliferation and fatal neurological diseases in neonatally infected gerbils

By using neonatal gerbils, we assessed the effect of ribavirin on the proliferation of Borna disease virus (BDV) in the brain. The intracranial inoculation of ribavirin reduced viral propagation in the acutely infected brain, resulting in protection from fatal neurological disorders. We found that the treatment with ribavirin markedly reduces the numbers of OX-42-positive microglial cells, but does not activate expression of Th1 cytokines, in BDV-infected gerbil brains. Our results suggested that ribavirin directly inhibits BDV replication and might be a potential tool for the treatment of BDV infection.

Implications for a regulated replication of Borna disease virus in brains of experimentally infected Lewis rats

The neurotropic Borna disease virus (BDV) causes typically a persistent virus infection of the central nervous system. In order to investigate whether an adapted virus replication contributes to BDV persistence in vivo, a fast and reliable real-time RT-PCR assay was constructed to quantify the amounts of leader-containing (leBDV) as a marker for virus replication, genomic (vBDV) and nucleoprotein-(BDV-N +ssRNA)-specific RNA. Therefore, leBDV, vBDV and BDV-N +ssRNA values were determined in experimentally infected Lewis rats between 14 and 90 days post infection (dpi). Surprisingly low leBDV values were found compared to vBDV and the abundantly expressed BDV-N transcripts. vBDV multiplied only in the acute phase of infection followed by constant expression until 90 dpi. Ratios of vBDV to leBDV were 401:1 at 14 dpi and diminished to 209:1 at 90 dpi, indicating a regulated co-expression of replicative intermediates as a potential prerequisite for viral persistence.

Infection of the enteric nervous system by Borna disease virus (BDV) upregulates expression of Calbindin D-28k

Borna disease virus (BDV) is a neurotropic agent infecting distinct neuronal subpopulations in the central nervous system of various mammalian species possibly including humans. Horses, a major natural host for BDV, show gastrointestinal dysfunctions besides characteristic neurological symptoms. Therefore, we hypothesized that enteric neurons may be targets of BDV replication. The presence of BDV-specific antigen in subpopulations of the ENS was investigated. Four-week-old Lewis rats were infected intracerebrally and sacrificed 4–14 weeks post infection (p.i.). BDV-immunoreactive neurons were found in submucous and myenteric neurons of the proximal colon. Fourteen weeks p.i., the proportion of BDV-positive neurons was 44 ± 17 and 24 ± 7% in the submucous and myenteric plexus, respectively. The majority of BDV-positive myenteric neurons showed immunoreactivity for choline acetyltransferase. Expression of Calbindin D-28k (CALB) was found in 96% of submucous and 67% of myenteric BDV-immunoreactive neurons. Additionally, the number of CALB-immunoreactive neurons was significantly higher in the myenteric plexus of infected rats compared to controls. These data indicate that BDV infects specific subpopulations of enteric neurons. Therefore, the ENS might serve as a site for BDV replication and as an immunoprivileged reservoir for BDV. In addition, upregulation of CALB in neurons of the myenteric plexus is probably induced during BDV-infection.

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.

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.

Borna Disease Virus Replication in Organotypic Hippocampal Slice Cultures from Rats Results in Selective Damage of Dentate Granule Cells

In the hippocampus of Borna disease virus (BDV)-infected newborn rats, dentate granule cells undergo progressive cell death. BDV is noncytolytic, and the pathogenesis of this neurodevelopmental damage in the absence of immunopathology remains unclear. A suitable model system to study early events of the pathology is lacking. We show here that organotypic hippocampal slice cultures from newborn rat pups are a suitable ex vivo model to examine BDV neuropathogenesis. After challenging hippocampal slice cultures with BDV, we observed a progressive loss of calbindin-positive granule cells 21 to 28 days postinfection. This loss was accompanied by reduced numbers of mossy fiber boutons when compared to mock-infected cultures. Similarly, the density of dentate granule cell axons, the mossy fiber axons, appeared to be substantially reduced. In contrast, hilar mossy cells and pyramidal neurons survived, although BDV was detectable in these cells. Despite infection of dentate granule cells 2 weeks postinfection, the axonal projections of these cells and the synaptic connectivity patterns were comparable to those in mock-infected cultures, suggesting that BDV-induced damage of granule cells is a post-maturation event that starts after mossy fiber synapses are formed. In summary, we find that BDV infection of rat organotypic hippocampal slice cultures results in selective neuronal damage similar to that observed with infected newborn rats and is therefore a suitable model to study BDV-induced pathology in the hippocampus.

Transgenic mice expressing the nucleoprotein of Borna disease virus in either neurons or astrocytes: decreased susceptibility to homotypic infection and disease.

The nucleoprotein (N) of Borna disease virus (BDV) is the major target of the disease-inducing antiviral CD8 T-cell response in the central nervous system of mice. We established two transgenic mouse lines which express BDV-N in either neurons (Neuro-N) or astrocytes (Astro-N). Despite strong transgene expression, neurological disease or gross behavioral abnormalities were not observed in these animals. When Neuro-N mice were infected as adults, replication of BDV was severely impaired and was restricted to brain areas with a low density of transgene-expressing cells. Notably, the virus failed to replicate in the transgene-expressing granular and pyramidal neurons of the hippocampus (which are usually the preferred host cells of BDV). When Neuro-N mice were infected within the first 5 days of life, replication of BDV was not suppressed in most neurons, presumably because the onset of transgene expression in the brain occurred after these cells became infected with BDV. Astro-N mice remained susceptible to BDV infection, but they were resistant to BDV-induced neurological disorder. Unlike their nontransgenic littermates, Neuro-N mice with persistent BDV infection did not develop neurological disease after immunization with a vaccinia virus vector expressing BDV-N. In contrast to the situation in wild-type mice, this treatment also failed to induce N-specific CD8 T cells in the spleens of both transgenic mouse lines. Thus, while resistance to BDV infection in N-expressing neurons appeared to result from untimely expression of a viral nucleocapsid component, the resistance to BDV-induced neuropathology probably resulted from immunological tolerance.

Borna disease virus induces acute fatal neurological disorders in neonatal gerbils without virus- and immune-mediated cell destructions

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that is known to cause neurological disturbances in various animal species. Our previous experiment demonstrated that neonate gerbils develop an acute fatal neurological disease following infection with BDV (Watanabe et al., 2001, Virology 282, 65–76). The study suggested that BDV directly causes functional damage of neuronal cells resulting in the lethal disorder in neonatal gerbils. To extend this finding, we examined whether BDV can induce neurological diseases in the absence of virus- and immune-mediated cell destruction, by using cyclosporine A (CsA)-treated neonatal gerbils. Although CsA completely suppressed specific antibody production and brain inflammation in the infected gerbil brains, the fatal neurological disorder was not inhibited by the treatment. Furthermore, we demonstrated that CsA treatment significantly decreased brain levels of cytokines, except interleukin (IL)-1β, in the infected gerbils. These results suggested that BDV replication, as well as brain cytokines, at least IL-1β, rapidly induces fatal disturbances in gerbil brain. We demonstrate here that BDV exhibits a unique neuropathogenesis in neonatal gerbil that may be pathologically and immunologically different from those in two other established rodent models, rats and mice. With this novel rodent model of virus infection it should be possible not only to examine acute neurological disturbances without severe neuroanatomical and immunopathological alterations but also to analyze molecular and cellular damage by virus replication in the central nervous system.

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.