Saori Yoshino

Glial expression of Borna disease virus phosphoprotein induces behavioral and neurological abnormalities in transgenic mice

One hypothesis for the etiology of behavioral disorders is that infection by a virus induces neuronal cell dysfunctions resulting in a wide range of behavioral abnormalities. However, a direct linkage between viral infections and neurobehavioral disturbances associated with human psychiatric disorders has not been identified. Here, we show that transgenic mice expressing the phosphoprotein (P) of Borna disease virus (BDV) in glial cells develop behavioral abnormalities, such as enhanced intermale aggressiveness, hyperactivity, and spatial reference memory deficit. We demonstrate that the transgenic brains exhibit a significant reduction in brain-derived neurotrophic factor and serotonin receptor expression, as well as a marked decrease in synaptic density. These results demonstrate that glial expression of BDV P leads to behavioral and neurobiological disturbances resembling those in BDV-infected animals. Furthermore, the lack of reactive astrocytosis and neuronal degeneration in the brains indicates that P can directly induce glial cell dysfunction and also suggests that the transgenic mice may exhibit neuropathological and neurophysiological abnormalities resembling those of psychiatric patients. Our results provide a new insight to explore the relationship between viral infections and neurobehavioral disorders.

Impaired development of the cerebellum in transgenic mice expressing the immediate-early protein IE180 of pseudorabies virus

Pseudorabies virus (PRV) infection in animals other than its natural host almost always gives rise to fatal diseases in the central nervous system as a result of infection of peripheral neurons and subsequently to the brain. PRV immediate-early protein (IE180) activates transcription of the PRV early and late genes, and other viral and cellular genes, and represses its own transcription. To examine specific effects of IE180 in neuropathogenicity, we have generated four transgenic mouse lines expressing IE180 in a tetracycline-regulated system. In the transgenic mouse lines, cerebellar symptoms such as ataxic gait, tremor and motor discoordination were observed. Histopathology of the cerebella in the transgenic mouse lines showing severe symptoms was remarkable for a failure of layer formation and a reduction in cerebellar size. These findings suggest that IE180 affects the cascade of gene expression for development of the murine cerebellum, resulting in the impairment of the cerebellar development and differentiation.

Cerebellar pathology in transgenic mice expressing the pseudorabies virus immediate‐early protein IE180

Pseudorabies virus is an alphaherpesvirus causing fatal neurological diseases in animals. Pseudorabies virus carries a gene encoding immediate‐early (IE) protein IE180, which controls the transcription of other viral and host cell genes. Previously, we reported that transgenic expression of IE180 in mice causes severe ataxia and cerebellar deformity. Here we identified profound abnormalities in adult IE180 transgenic mice, including malpositioning of Purkinje cells (PCs), granule cells (GCs) and Bergmann glia (BG), impaired dendritogenesis and synaptogenesis in PCs, disoriented BG fibers, absence of molecular layer interneurons, and increased apoptosis of neurons and glia. In accordance with the cellular defects, we found the expression of IE180 in PCs, GCs and astrocytes during cerebellar development. We next examined transgenic mice expressing truncated IE180 mutants: dlN132 lacking the acidic transcriptional active domain, dlC629 lacking the nuclear localization signal and dlC1081 having all known domains but lacking the carboxyl‐terminal sequence. Despite similar expression levels of the transgenes, ataxia and cerebellar defects were only manifested in the dlC1081 transgenic mice but their phenotypes were milder compared with the IE180 transgenic mice. In the dlC1081 transgenic mice, cerebellar neurons and glia were normally positioned but cerebellar size was severely reduced due to GC deficits. Interestingly, dlC1081 was mainly expressed in the GCs with low expression in a few BG. Taken together, the present findings clarified a causal relationship between cerebellar pathology and cellular expression of IE180, and further afforded an experimental insight into different symptomatic severity as a consequence of different cellular defects caused by such cytotoxic viral agents.

Analysis of regulatory functions for the region located upstream from the latency-associated transcript (LAT) promoter of pseudorabies virus in cultured cells.

The latency-associated transcript (LAT) promoter of pseudorabies virus (PrV) is unique among the many promoters of the viral genome in that it remains active during the latent state. The regulatory mechanism of PrV LAT gene expression is complex and different between latency and lytic infection of cultured cells. Although two different sequences, LAP1 and LAP2, are thought to be involved in LAT gene expression, the function of the upstream region of the LAT promoter (LAP1 and LAP2) remains an enigma, even in cultured cells. To analyze the function of the upstream region, it is necessary to examine the effects of the upstream sequence on LAT gene expression in the absence of other viral proteins. Transient expression assays were performed by employing a series of reporter plasmids in which various sequences upstream of the LAT promoter (from nucleotide positions -592 to +423 relative to the transcriptional start site of the large latency transcript (LLT)) were linked to the chloramphenicol acetyltransferase (CAT) gene in cells of neuronal and non-neuronal origin. We identified a region (from nucleotide positions -3606 to -1386) that was capable of repressing the LAT promoter activity in Vero cells by analyzing CAT gene expression of the series of reporter plasmids. This effect was not observed in Neuro-2a cells. We have also shown that the LAT promoter activity of the reporter plasmid containing the upstream region was repressed by the immediate-early gene product IE180 in Vero cells, but not in Neuro-2a cells. These results suggest that the upstream region of the LAT promoter may have a role in repressing LAT gene expression in cultured non-neuronal cells.

Inhibition of pseudorabies virus replication by a dominant-negative mutant of early protein 0 expressed in a tetracycline-regulated system

Pseudorabies virus (PRV) early protein 0 (EP0) consisting of 410 amino acids is a transactivator of viral genes. A mutant consisting of amino acids 1-113 exhibits dominant-negative properties. In order to assess the antiviral potential of the EP0 mutant, Vero cells were transformed with the EP0 mutant gene expressed in a tetracycline-regulated system. The transformed cell lines showed marked resistance to PRV infection when expression of the EP0 mutant gene was induced. In the transformed cell line infected with PRV, synthesis of the immediate-early protein (IE180) and of EP0 was inhibited, whereas the levels of IE and EP0 messenger RNA (mRNA) were not decreased, as compared with those of the control cell line. The present results suggest that the EP0 mutant may not alter the efficiency of the viral gene transcription but rather translation efficiency of the viral mRNA.

A pan-specific promoter activity of the 213bp segment of the pseudorabies virus early protein 0 gene in transgenic mice

Summary. Pseudorabies virus (PrV) early protein 0 (EP0) is a transactivator that plays important roles in the viral gene expression. To examine a promoter regulatory element for the EP0 gene expression in vivo, we have generated two transgenic mouse lines expressing the EP0 gene under the control of a 213bp 5′-flanking sequence of the EP0 gene. To analyze the tissue specificity of transgene expression, mRNA of the EP0 gene was monitored in various tissues from the transgenic mice by the reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis. The EP0 gene expression was demonstrated in all tissues tested by the RT-PCR analysis. These tissues included skin, muscles (skeletal and heart), lung, liver, spleen, small intestine, kidney, brain and testis. These results indicated that the 213bp sequence of the 5′-flanking region of the EP0 gene is capable of driving expression of the EP0 gene in vivo and the promoter is pan-specific.

The pseudorabies virus immediate-early promoter directs neuronal tissue-specific expression in transgenic mice

Summary. Pseudorabies virus (PRV) immediate-early (IE) gene product is required for expression of the viral early and late genes as a transactivator. The IE gene is expressed as the first gene among the viral genes after the infection. To examine the activity of the IE promoter in vivo, we have generated transgenic mice expressing transgenes under the control of the IE promoter. To analyze the tissue specificity of the transgene expression, mRNA of the transgene was monitored in various tissues from the transgenic mice by reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis. A strong transgene expression was observed in the neuronal tissues by the RT-PCR analysis. These neuronal tissues included cerebrum, cerebellum and trigeminal nerve. Although the PCR product was hardly detected in other tissues by the RT-PCR analysis, specific PCR bands were detected in multiple organs (skin, skeletal muscles, heart muscles, lung, liver, spleen, small intestine and kidney) by Southern blot analysis using the RT-PCR products. These results indicate that although the IE promoter acts as a pan-specific promoter in vivo, it is capable of driving a high level of transgene expression in neuronal tissues.