Nucleoprotein (P40) Binding to 5HT2C Receptors (5HT2CR) is the Key Point in the Pathogenesis of BoDV-1-Infected Hosts.

Nucleoprotein (P40) is one of the most important proteins of Borna disease virus 1 (BoDV-1), but which proteins it would bind to in the pathogenesis of BoDV-1-infected hosts is unknown. We used lentivirus LV5-P40 overexpressing P40 to infect primary hippocampal neurons and characterized the interactome of P40 with co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS) analysis. These interacting protein partners revealed the pathogenesis of BoDV-1-infected hosts. We also show for the first time that P40 interacts with 5HT2CR in rat neurons, which may be the molecular basis leading to neuropsychiatric diseases such as anxiety disorders and behavioral abnormalities after BoDV-1 infection of hosts.

No evidence for European bats serving as reservoir for Borna disease virus 1 or other known mammalian orthobornaviruses.

BACKGROUND: The majority of emerging infectious diseases are zoonotic in nature and originate from wildlife reservoirs. Borna disease, caused by Borna disease virus 1 (BoDV-1), is an infectious disease affecting mammals, but recently it has also been shown to cause fatal encephalitis in humans. The endemic character of Borna disease points towards a nature-bound reservoir, with only one shrew species identified as reservoir host to date. Bats have been identified as reservoirs of a variety of zoonotic infectious agents. Endogenous borna-like elements in the genome of certain bat species additionally point towards co-evolution of bats with bornaviruses and therefore raise the question whether bats could serve as a potential reservoir of orthobornaviruses. METHODS: Frozen brain samples (n = 257) of bats of seven different genera from Germany were investigated by orthobornaviral RT-PCR. Additionally, tissue slides of formalin-fixed paraffin-embedded material of a subset of these bats (n = 140) were investigated for orthobornaviral phosphoprotein by immunohistochemistry. RESULTS: The brain samples were tested by RT-PCR without any evidence of orthobornavirus specific amplicons. Immunohistochemistry revealed a faint immunoreaction in 3/140 bats but with an untypical staining pattern for viral antigen. CONCLUSIONS: RT-PCR-screening showed no evidence for orthobornaviral RNA in the investigated bats. However, immunohistochemistry results should be investigated further to elucidate whether the reaction might be associated with expressed endogenous bornaviral elements or other so far unknown bornaviruses.

Proteomics computational analyses suggest that the bornavirus glycoprotein is a class III viral fusion protein (gamma penetrene).

BACKGROUND: Borna disease virus (BDV) is the type member of the Bornaviridae, a family of viruses that induce often fatal neurological diseases in horses, sheep and other animals, and have been proposed to have roles in certain psychiatric diseases of humans. The BDV glycoprotein (G) is an extensively glycosylated protein that migrates with an apparent molecular mass of 84,000 to 94,000 kilodaltons (kDa). BDV G is post-translationally cleaved by the cellular subtilisin-like protease furin into two subunits, a 41 kDa amino terminal protein GP1 and a 43 kDa carboxyl terminal protein GP2. RESULTS: Class III viral fusion proteins (VFP) encoded by members of the Rhabdoviridae, Herpesviridae and Baculoviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Proteomics computational analyses suggest that the structural/functional motifs that characterize class III VFP are located collinearly in BDV G. Structural models were established for BDV G based on the post-fusion structure of a prototypic class III VFP, vesicular stomatitis virus glycoprotein (VSV G). CONCLUSION: These results suggest that G encoded by members of the Bornavirdae are class III VFPs (gamma-penetrenes).

[Expressing borna disease virus phosphoprotein in PC-12 cell and its influence on the cell proliferation].

OBJECTIVE: To establish stable expressing system of Borna disease virus (BDV) phosphoprotein in PC-12 cells, and then study its influence on cell proliferation of PC-12 cells. METHOD: An expression plasmid with green fluorescence protein was cloned and identified to express BDV phosphoprotein. Cultured PC-12 cell was transfected with the recombinant plasmid by positive ion lipidsome method. Fluorescence microscopy was used to detect the expression of phosphoprotein in PC-12 cells, then G418 was added into cell culture medium to kill cells without recombinant plasmid. We performed reverse transcriptase polymerase chain reaction (RT-PCR) in the 10th generation of treated cells to examine the expression of BDV phosphoprotein. The proliferation of treated cells and control cells was examined by methyl thiazolyl tetrazolium assay (MT). RESULT: The recombinant plasmid was confirmed to be able to express BDV phosphoprotein and green fluorescence protein by both fluorescence microscopy and RT-PCR. BDV phosphoprotein expressed in PC-12 cell inhibited cell proliferation. CONCLUSION: We established a stable expressing system of BDV phosphoprotein in PC-12 cell. This cell model can be used to study the effect of BDV phosphoprotein on the centre nervous system without exposure to live virus.

Crystal structure of the Borna disease virus matrix protein (BDV-M) reveals ssRNA binding properties.

Borna disease virus (BDV) is a neurotropic enveloped RNA virus that causes a noncytolytic, persistent infection of the central nervous system in mammals. BDV belongs to the order Mononegavirales, which also includes the negative-strand RNA viruses (NSVs) Ebola, Marburg, vesicular stomatitis, rabies, mumps, and measles. BDV-M, the matrix protein (M-protein) of BDV, is the smallest M-protein (16.2 kDa) among the NSVs. M-proteins play a critical role in virus assembly and budding, mediating the interaction between the viral capsid, envelope, and glycoprotein spikes, and are as such responsible for the structural stability and individual form of virus particles. Here, we report the 3D structure of BDV-M, a full-length M-protein structure from a nonsegmented RNA NSV. The BDV-M monomer exhibits structural similarity to the N-terminal domain of the Ebola M-protein (VP40), while the surface charge of the tetramer provides clues to the membrane association of BDV-M. Additional electron density in the crystal reveals the presence of bound nucleic acid, interpreted as cytidine-5'-monophosphate. The heterologously expressed BDV-M copurifies with and protects ssRNA oligonucleotides of a median length of 16 nt taken up from the expression host. The results presented here show that BDV-M would be able to bind RNA and lipid membranes simultaneously, expanding the repertoire of M-protein functionalities.

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.

Structural comparisons of the nucleoprotein from three negative strand RNA virus families.

Structures of the nucleoprotein of three negative strand RNA virus families, borna disease virus, rhabdovirus and influenza A virus, are now available. Structural comparisons showed that the topology of the RNA binding region from the three proteins is very similar. The RNA was shown to fit into a cavity formed by the two distinct domains of the RNA binding region in the rhabdovirus nucleoprotein. Two helices connecting the two domains characterize the center of the cavity. The nucleoproteins contain at least 5 conserved helices in the N-terminal domain and 3 conserved helices in the C-terminal domain. Since all negative strand RNA viruses are required to have the ribonucleoprotein complex as their active genomic templates, it is perceivable that the (5H+3H) structure is a common motif in the nucleoprotein of negative strand RNA viruses.

Oligomerization and assembly of the matrix protein of Borna disease virus.

The matrix protein M of Borna disease virus (BDV) is a constituent of the viral envelope covering the inner leaflet of the lipid bilayer. BDV-M was expressed as recombinant protein in Escherichia coli, purified to homogeneity and structurally analyzed. Recombinant M (i) forms non-covalently bound multimers with a Stoke's radius of 35 Angstroms estimated by size exclusion chromatography, (ii) consists of tetramers detected by analytical ultracentrifugation, and (iii) appears by electron microscopy studies as tetramers with the tendency to assemble into high molecular mass lattice-like complexes. The structural features suggest that BDV-M possesses a dominant driving force for virus particle formation.

Inhibition of Borna disease virus-mediated cell fusion by monoclonal antibodies directed against the viral glycoprotein.

Borna disease virus-infected Vero cells express on their surface the major viral glycoprotein which mediates cell fusion after low pH treatment. This fusion event can be inhibited by monoclonal antibodies (mAbs) generated from chronically BDV-infected rats boosted with a recombinant vaccinia virus expressing the gp94 glycoprotein of BDV. Analysis of mAbs suggests specificity for the 43-kD C-terminal furin cleavage product of gp94 and provides evidence for the recognition of a conformational epitope. The results confirm and extend earlier findings on the presence of the gp43 protein on the surface of BDV-infected cells and its specific role in cell fusion.

Crystal structure of the borna disease virus nucleoprotein.

Borna disease virus (BDV) causes an infection of the central nervous system in a wide range of vertebrates, which can fatally progress to an immune-mediated disease, called Borna disease. BDV is a member of the Mononegavirales, which also includes the highly infectious measles and Ebola viruses. The viral nucleoproteins are central to transcription, replication, and packaging of the RNA genome. We present the X-ray structure of the BDV nucleoprotein determined at 1.76 A resolution. The structure reveals a novel fold, organized into two distinct domains, and an assembly into a planar homotetramer. Surface potential calculations strongly support an RNA binding model with the RNA wrapping around the outside of the tetramer, although a positively charged central channel in the tetramer could fit single-stranded RNA in an alternative binding mode. This first structure of an RNA virus nucleoprotein provides a paradigmatic model for RNA packaging and replication of single-stranded RNA viruses.

Enhanced neurovirulence of borna disease virus variants associated with nucleotide changes in the glycoprotein and L polymerase genes.

Borna disease virus (BDV) infection produces a variety of clinical diseases, from behavioral illnesses to classical fatal encephalitis (i.e., Borna disease [BD]). Since the genomes of most BDV isolates differ by less than 5%, host factors are believed responsible for much of the reported variability in disease expression. The contribution of BDV genomic differences to variation in BD expression is largely unexplored. Here we compared the clinical outcomes of rats infected with one of two related BDV variants, CRP3 or CRNP5. Compared to rats inoculated with CRP3, adult and newborn Lewis rats inoculated with CRNP5 had more severe and rapidly fatal neurological disease, with increased damage to the hippocampal pyramidal neurons and rapid infection of brain stem neurons. To identify possible virus-specific contributions to the observed variability in disease outcome, the genomes of CRP3 and CRNP5 were sequenced. Compared to CRP3, there were four nucleotide changes in the CRNP5 variant, two each in the G protein and in the L polymerase, resulting in four amino acid changes. These results suggest that small numbers of genomic differences between BDV variants in the G protein and/or L polymerase can contribute to the variability in BD outcomes.

Crystallization and preliminary X-ray analysis of the matrix protein of Borna disease virus.

The matrix protein M of Borna disease virus (BDV) is associated with the inner viral membrane and is thought to be a mediator between the nucleocapsid and the lipid-containing envelope in stabilizing the virus shape. The full-length BDV-M gene encoding a 16 kDa protein was expressed in Escherichia coli. M was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. The crystals of M belong to the space group I432, with unit-cell parameters a = b = c = 144.6 A, and diffract to 3.1 A.

Molecular and cellular biology of Borna disease virus infection.

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that causes neurobehavioral disorders in a wide variety of warm-blooded animals. Recent evidence has revealed that BDV uses a unique strategy in its transcription and replication and directly affects cellular functions of infected central nervous systems. BDV research will provide new insights not only into the biology of neurotropic RNA virus but also into neuropsychiatry.

Open reading frame III of borna disease virus encodes a nonglycosylated matrix protein.

The open reading frame III of Borna disease virus (BDV) codes for a protein with a mass of 16 kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N- nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous proteins of other members of the Mononegavirales order.

A naturally processed rat major histocompatibility complex class I-associated viral peptide as target structure of borna disease virus-specific CD8+ T cells.

The first naturally processed peptide synthesized by a virus and recognized by classical CD8(+) T cells in association with the RT1.A(l) major histocompatibility complex class I molecule of the Lewis rat is reported. Borna disease virus-specific CD8(+) T cells recognize syngeneic target cells pulsed with peptides extracted from Borna disease virus-infected cells. The predicted peptide sequence ASYAQMTTY from the viral p40 protein coeluted with the cytotoxic T-lymphocyte-reactive fraction was identified among natural ligands by tandem mass spectrometry. Numerous naturally processed peptides derived from intracellular bacteria, viruses, or tumors and recognized by CD8(+) T cells of man and mice are known, leading to a better understanding of cellular immune mechanisms against pathogens in these two species. In contrast, for the rat little information exists with regard to the function and role of CD8(+) T cells as part of their cellular immune defense system. This first naturally processed viral epitope in the rat contributes to the understanding of the rat cellular immune response and might trigger the identification of more cytotoxic T-lymphocyte epitopes in this animal.

Borna disease virus: new aspects on infection, disease, diagnosis and epidemiology.

A 'disease of the head' affecting horses, as described in the 17th Century is now known as Borna disease. Research over the past 100 years has established that the aetiological agent, Borna disease virus (BDV), is an unsegmented, single- and negative-stranded, enveloped ribonucleic acid (RNA) virus which represents the family Bornaviridae in the order Mononegavirales. The virus exists world-wide in horses, sheep, cattle, cats, dogs and ostriches. The infection can be fatal, but the majority of carriers are persistently infected without showing symptoms. The association with psychiatric diseases in humans led to an international explosion of research on BDV, with centres established in Germany, the United States of America and Japan. Experimental infections of tree shrews and rats served to examine the effects of persistent and overt disease, most excitingly, virus-induced behavioural changes, and emotional and learning deficits. This 'emerging' virus infection shows complex pathogenetic mechanisms in the nervous system, but also spreads through myelo-monocytic cells. Diagnosis can be made serologically, but detection of antigen markers in peripheral white blood cells, combined with nucleic acid amplification is more profitable. Comparative RNA studies reveal an unusually high genetic homology of viruses. Isolates recovered from humans and equines suggest species-specificity. Vaccination is not an advisable strategy, but antiviral therapy, especially with amantadine sulphate, promises efficacy in human mood disorders, and is effective in vitro. Infections with BDV follow a vulnerability principle to cause disease. Although cross-species transmission of this commensal virus has not been proven, zoonotic aspects of BDV should be carefully considered.

Borna disease virus infection in domestic cats: evaluation by RNA and antibody detection.

Borna disease virus (BDV) infection has been suggested to cause spontaneous neurological disease in cats referred to as staggering disease. However the evaluation of BDV infection in neurologically asymptomatic cats remained unclear. In the present study, BDV infected, asymptomatic cats in Tokyo were surveyed both by the presence of plasma antibodies against BDV-p24 and -p40 and by RNA detection in peripheral blood mononuclear cells. Seven of 32 domestic cats (21.9%) were serologically or genetically judged to be BDV-infected. Six cats were positive for anti-BDV antibody and two cats were positive for BDV RNA. Within the 2 RNA-positive cats, only one was positive for anti-BDV antibodies. Furthermore, the findings of anti-BDV-p40 and anti-BDV-p24 antibody-positive cats did not completely overlap. These results suggest that there are neurologically asymptomatic domestic cats infected with BDV present in the Tokyo area.

Neutralization of borna disease virus depends upon terminal carbohydrate residues (alpha-D-man, beta-D-GlcNAc) of glycoproteins gp17 and gp94.

Borna disease virus (BDV) is an enveloped, nonsegmented, negative-stranded RNA virus that causes infections of the brain in a wide range of animal species and man. The third open reading frame codes for a protein of 17 kD (gp17) that is N-glycosylated and contains terminal alpha-D-mannose and N-acetyl-beta-D-glucosamine residues. Rat sera raised against these carbohydrates (anti-sugar antisera) show high in vitro neutralization activity and were capable of precipitating BDV. The neutralizing capacity of sera derived from experimentally BDV-infected rabbits, in turn, decreased after adsorption with those carbohydrates. They partially inhibited infection of primary young rabbit brain cells in a dose-dependent manner. Furthermore, the anti-sugar antisera recognized a second virus-specific glycoprotein with an apparent molecular mass of 94 kD (gp94), providing indirect evidence that gp94 is involved in virus adsorption and/or entry into cells. Neutralization of BDV comprises a complex event and, as shown for the first time, involves the carbohydrate residues of both glycoproteins of BDV.