Borna disease virus phosphoprotein binds a neurite outgrowth factor, amphoterin/HMG-1.

The Borna disease virus (BDV) p24 phosphoprotein is an abundant protein in BDV-infected cultured cells and animal brains. Therefore, there is a possibility that binding of the p24 protein to cellular factor(s) induces functional alterations of infected neural cells in the brain. To identify a cellular protein(s) that interacts with BDV p24 protein, we performed far-Western blotting with extracts from various cell lines. Using recombinant p24 protein as a probe, we detected a 30-kDa protein in all cell lines examined. Binding between the 30-kDa and BDV p24 proteins was also demonstrated using BDV p24 affinity and ion-exchange chromatography columns. Microsequence analysis of the purified 30-kDa protein revealed that its N terminus showed complete homology with rat amphoterin protein, which is a neurite outgrowth factor abundant in the brain during development. Mammalian two-hybrid and immunoprecipitation analyses also confirmed that amphoterin is a specific target for the p24 protein in vivo. Furthermore, we showed that infection by BDV, as well as purified p24 protein in the medium, significantly decreased cell process outgrowth of cells grown on laminin, indicating the functional inhibition of amphoterin by interaction with the p24 protein. Immunohistochemical analysis revealed decreased levels of amphoterin protein at the leading edges of BDV-infected cells. Moreover, the expression of the receptor for advanced glycation end products, of which the extracellular moiety is a receptor for amphoterin, was not significantly activated in BDV-infected cells during the process of extension, suggesting that the secretion of amphoterin from the cell surface is inhibited by the binding of the p24 protein. These results suggested that BDV infection may cause direct damage in the developing brain by inhibiting the function of amphoterin due to binding by the p24 phosphoprotein.

Monoclonal antibodies against topoisomerase I suppressed DNA relaxation and HIV-1 cDNA synthesis.

Human immunodeficiency virus type 1 (HIV-1) virion is known to carry a number of cellular components including cellular topoisomerase I. Previously, we have demonstrated that topoisomerase I enhances HIV-1 cDNA synthesis in reverse transcription (RT) assays in vitro. In the present study, we have produced six monoclonal antibodies (MAbs) against human topoisomerase I. The MAbs suppressed nicking/closing of supercoiled DNA and cDNA synthesis in an endogenous reverse transcription (ERT) assay using a detergent-disrupted HIV-1 virion. Thus, the results suggest that topoisomerase I plays an important role in RNA-directed DNA polymerization.

Isolation of Borna disease virus from human brain tissue.

Serological and molecular epidemiological studies indicate that Borna disease virus (BDV) can infect humans and is possibly associated with certain neuropsychiatric disorders. We examined brain tissue collected at autopsy from four schizophrenic patients and two healthy controls for the presence of BDV markers in 12 different brain regions. BDV RNA and antigen was detected in four brain regions of a BDV-seropositive schizophrenic patient (P2) with a very recent (2 years) onset of disease. BDV markers exhibited a regionally localized distribution. BDV RNA was found in newborn Mongolian gerbils intracranially inoculated with homogenates from BDV-positive brain regions of P2. Human oligodendroglia (OL) cells inoculated with brain homogenates from BDV-positive gerbils allowed propagation and isolation of BDVHuP2br, a human brain-derived BDV. Virus isolation was also possible by transfection of Vero cells with ribonucleoprotein complexes prepared from BDV-positive human and gerbil brain tissues. BDVHuP2br was genetically closely related to but distinct from previously reported human- and animal-derived BDV sequences.

Two proline-rich nuclear localization signals in the amino- and carboxyl-terminal regions of the Borna disease virus phosphoprotein.

Borna disease virus (BDV) uses a unique strategy of replication and transcription which takes place in the nucleus, unlike other known, nonsegmented, negative-stranded RNA viruses of animal origin. In this process, viral constituents necessary for replication must be transported to the nucleus from the cytoplasm. We report here the evidence that BDV P protein, which may play an important role in viral replication and transcription, is transported into the nucleus in the absence of other viral constituents. This transportation is accomplished by its own nuclear localization signals (NLSs), which are present in both N-terminal (29PRPRKIPR36) and C-terminal (181PPRIYPQLPSAPT193) regions of the protein. These two NLSs can function independently and both have several Pro residues as key amino acids.

Nuclear targeting activity associated with the amino terminal region of the Borna disease virus nucleoprotein.

The Borna disease virus (BDV) replicates in the nucleus. The viral p40 protein (N), which is found abundantly in the nucleus in BDV-infected cells, may play an important role in virus replication. To analyze the amino acid residues involved in the nuclear targeting of BDV N, a series of eukaryotic expression plasmids encoding deletion mutants of N was constructed and transfected into COS-7 cells. In indirect immunofluorescence assays with a rabbit anti-BDV N antiserum, wild-type N was located in the nucleus of transfected cells in the absence of other viral constituents. In contrast, mutants lacking the 13 NH2-terminal amino acid residues 1MPPKRRLVDDADA13 in common gave a cytoplasmic localization pattern. Similarly, a mutant with substitution of 4KRR6 by 4NSG6 was retained in the cytoplasm. Furthermore, a nonapeptide, 3PKRRLVDDA11, derived from the NH2-terminal region of N conferred nuclear targeting activity to beta-galactosidase, which normally resides in the cytoplasm. Thus, we have identified the nuclear targeting signal of the BDV N and narrowed it to the NH2-terminal region where 4KRR6 basic amino acid residues are located.

Protective effect of passive immunization against TNF-alpha in mice infected with Sendai virus.

TNF-alpha has been reported to be induced in mice infected with Sendai virus. We evaluated the role of TNF-alpha in the virus infection. TNF-alpha was induced locally in proportion to virus titers in the lung. The activity was correlated with suppression of body weight gain. Passive immunization against TNF-alpha improved body weight gain and ameliorated pneumonic lesions in infected mice, and prevented them from lethal infection, but lung virus induced emaciation, pneumonic lesions and death were mediated by TNF-alpha.

Amplification of a full-length Borna disease virus (BDV) cDNA from total RNA of cells persistently infected with BDV.

We have developed a novel reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify the full-length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RNA of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53-mer oligonucleotide primer, corresponding to the 5'-terminus of a putative 3'-leader sequence of the BDV RNA genome, for 2 hr at 42 C followed by 30 min at 55 C. PCR was performed in the presence of this 53-mer antigenomic primer and a 25-mer primer, corresponding to the 3'-terminus of the BDV antigenomic cDNA, by use of an rTth DNA polymerase with proof-reading activity. The amplified full-length BDV cDNA was detected in as little as 20 ng of total cellular RNA of MDCK/BDV. This RT-PCR method should be a useful technique to study the molecular quasispecies of BDV.

Sequence variability of Borna disease virus open reading frame II found in human peripheral blood mononuclear cells.

A cDNA fragment of the Borna disease virus (BDV) open reading frame II (ORF-II), which encodes a 24-kDa phosphoprotein (p24 [P protein]), was amplified from total RNA of peripheral blood mononuclear cells (PBMC) from three psychiatric inpatients. The amplified cDNA fragments were cloned, sequenced, and analyzed. A total of 15 clones, 5 from each patient, were studied. Intrapatient divergencies of the BDV ORF-II nucleotide sequence were 4.2 to 7.3%, 4.8 to 7.3%, and 2.8 to 7.1% for the three patients, leading to differences of 7.7 to 14.5%, 10.3 to 17.1%, and 6.0 to 16.2%, respectively, in the deduced amino acid sequence for BDV p24. Interpatient divergencies among the 15 clones were 5.9 to 12.7% at the nucleotide level and 12.8 to 28.2% at the amino acid level. Thus, in p24, BDV in human PBMC of the patients undergoes mutation at high rates in vivo. Additionally, we found that the nucleotide sequence of the 15 human BDV ORF-II cDNA clones differed from those of the horse strains V and He/80-1 by 4.2 to 9.3%. However, comparison of the consensus amino acid sequence deduced from the 15 human clones with those of the horse strains revealed no human-specific amino acid residue, suggesting that the BDV infecting humans may be related to that infecting horses.