Yuko Shoya

Isolation of Borna Disease Virus from Human Brain Tissue

ABSTRACT 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.

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

ABSTRACT 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.

Nucleolin and the Packaging Signal, ψ, Promote the Budding of Human Immunodeficiency Virus Type-1 (HIV-1)

Gag proteins of human immunodeficiency virus type 1 (HIV‐1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA‐binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV‐1 gag proteins, the packaging signal, ψ and nucleolin affect the budding of HIV‐1. Here we report that nucleolin enhances the release of HIV‐1 virions which contain ψ. Furthermore, nucleolin and gag proteins form a complex incorporated into virions, and nucleolin promotes the infectivity of HIV‐1. Our results suggest that an empty particle which contains neither nucleolin nor the genomic RNA is eliminated during the budding process, and this mechanism is beneficial for escape from the host immune response against HIV‐1.

Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type 1.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I-RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1.

Human topoisomerase I promotes HIV-1 proviral DNA synthesis: Implications for the species specificity and cellular tropism of HIV-1 infection

Although HIV type 1 (HIV-1) cannot efficiently replicate in simian cells, the mechanism(s) involved in the restriction of virus tropism remain unclear. To investigate this, we have focused on the identification of human cellular factors that can influence the infectivity of HIV-1 derived from African green monkey producer cells. Whereas the infectivity of HIV-1 derived from such cells was only 10–15% of that of human cell-derived virus, expression of human topoisomerase I in the African green monkey cells resulted in a 5-fold increase of the infectivity of progeny HIV-1 virions. Replacement of glutamate-236 and asparagine-237 of human topoisomerase I with the corresponding residues (aspartate and serine, respectively) of the African green monkey enzyme abolished this enhancement of HIV-1 infectivity. This positive effect of human topoisomerase I expression in the African green monkey producer cells seemed to result from the promotion of HIV-1 cDNA synthesis. Thus, human topoisomerase I plays an important role in HIV-1 replication and infectivity, and differences in the species specificity of HIV-1 infection can at least in part be attributed to differences in topoisomerase I activities.

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.

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.