Kei Amemiya

Interaction of the human polyomavirus, JCV, with human B-lymphocytes.

The human polyomavirus, JCV, is the causative agent of the central nervous system demyelinating disease progressive multifocal leukoencephalopathy (PML). The principal target of JCV infection in the central nervous system (CNS) is the myelinating oligodendrocyte. However, the site of JCV multiplication outside of the CNS and the mechanism by which virus gains access to the brain are not known. Recently, JCV infected B-lymphocytes have been demonstrated in PML patients in several lymphoid organs, in circulating peripheral lymphocytes, and in brain, suggesting a possible role of B-lymphocytes in the dissemination of virus to the brain. The experiments reported here were undertaken to understand more about the interactions of JCV with human B-lymphocytes. The data show that JCV is able to multiply in either Epstein-Barr virus transformed (EBV) or EBV negative human B cell lines resulting in production of infectious, progeny virions. In addition, nuclear proteins extracted from these B cells bind to similar nucleotides within the JCV regulatory region that are bound by nuclear proteins extracted from human fetal glial cells, the most susceptible host and principal target cell for JCV infection in vitro. It is not known, however, whether these DNA binding proteins from susceptible B cells and glial cells are similar.

Expression of multiple classes of the nuclear factor-1 family in the developing human brain: differential expression of two classes of NF-1 genes.

Nuclear factor-1 (NF-1) is a multifunctional protein that participates in both transcription and replication. NF-1 proteins exist as a family of proteins that share some common structural and functional features but also demonstrate organ and cell type specific expression. Based upon these characteristics, the family of NF-1 proteins is divided into four classes, A, B, C and D. Several NF-1 binding sites have been identified in the regulatory sequences of the human polyomavirus, JCV, which multiplies most efficiently in glial cells derived from human fetal brain. Nuclear proteins from these cultures bind specifically to these NF-1 sites. It is not known, however, which member(s) of the NF-1 family is expressed in cells susceptible to JCV infection. We have examined glial cells as well as HeLa cells, which are not permissive to JCV, for NF-1 expression. By RT-PCR analysis, all four classes of NF-1 are expressed in human fetal glial cells and HeLa cells. However, by Northern analysis the expression of class D gene is much higher in the glial cells than HeLa cells. Expression of the class C gene, first identified in HeLa cells as NF-1/CTF1, is barely detectable in glial cells but highly expressed in HeLa cells. The screening of cDNA libraries from two early human brain tissues resulted in the identification of a number of clones which appear to be related and belong to a single class of the NF-1 family, class D. Nucleotide sequence of one clone, designated NF-1/AT1, confirms this. The NF-1/AT1 protein was overexpressed in E coli and found to bind specifically to an NF-1 probe by gel shift analysis. Southern analysis of human fetal glial cells indicates that the NF-1/AT1 gene, class D, is derived from a different gene than NF-1/CTF1. These results suggest the possibility that genes or viruses, like JCV, which use NF-1 for their expression in human brain derived cells may preferentially use the NF-1 class D protein.

Evaluation of the role of cytokine activation in the multiplication of JC virus (JCV) in human fetal glial cells.

The human polyomavirus, JCV, is the etiologic agent of the fatal central nervous system demyelinating disease, progressive multifocal leukoencephalopathy. Progressive multifocal leukoencephalopathy occurs most frequently in patients with underlying immunosuppressive disorders and is the direct result of virus multiplication in oligodendrocytes, the myelin producing cell in the central nervous system. In this report we test the ability of cellular activation signals to modulate expression of the JCV genome in either transfected or infected human fetal glial cells. In addition, we analyze the binding of nuclear proteins isolated from untreated and cytokine treated human fetal glial cells to transcription factor binding sites in the JCV regulatory region. In contrast to the effects of cellular activation on the expression of the HIV-1 promoter in these cells, none of the cellular activators tested increased expression of JCV. The cytokine, TNF-alpha, increased binding of NF kappa B (p50/p65) to a JC NF kappa B site but did not modulate the binding of nuclear proteins to the overlapping NF-1/AP1 region of the JCV enhancer. When taken together these results suggest that the response of JCV to cellular activation signals may be fundamentally different from the response of HIV-1 to these signals in human fetal glial cells and that the JC NF kappa B site may not be required for JCV gene expression or multiplication in vivo.

Conserved sequence elements upstream and downstream from the transcription initiation site of the Caulobacter crescentus rrnA gene cluster

The nucleotide sequence and in vivo transcription start sites for rrnA, one of the two rRNA gene clusters of the eubacterium Caulobacter crescentus, have been determined. Two transcription start sites, a major and minor, for the rRNA gene cluster are located more than 700 nucleotides upstream from the 16 S rRNA gene. Transcription was detected from only the major start site in swarmer cells. But after the swarmer-to-stalked cell transition, transcription was detected from both rRNA start sites and continued throughout the developmental cell cycle when cells were grown in minimal medium. On the other hand, transcription from only the major start site was detected in cells growing in a complex medium. A small open reading frame was found upstream from the rRNA gene transcription start sites and was followed by an inverted repeat sequence. No sequence homology was found between the major rRNA gene transcription start site and the Escherichia coli sigma 70 promoters or the consensus sequence elements reported for C. crescentus fla promoters. However, there were two areas of homology when the major rRNA gene promoter was compared to the nucleotide sequence of the C. crescentus trpFBA promoter. There was a 12 nucleotide sequence centered around the -10 region of both promoters that was closely homologous. In addition, immediately downstream from the transcription start there was a sequence element that was identical in both promoters. These nucleotide sequence elements were not in the temporally expressed fla promoters of C. crescentus.

Cell Cultures from Human Fetal Brain Provide a Model for HIV-1 Persistence and Reactivation in the Central Nervous System

Cell cultures derived from human fetal tissues have been used for many years for the laboratory diagnosis of viral infections. In 1971, cultures prepared from human fetal brain were used in the isolation of a human polyomavirus, JCV, which was identified as the etiologic agent for the demyelinating disease, progressive multifocal leukoencephalopathy.1,2 Since JCV infected the oligodendrocyte in the brain, it was thought that JCV also infected the oligodendrocyte or its precursor cell in cultures from fetal brain.3,4 Laboratory investigations of the lineage of fetal brain-derived cells revealed that these precursor cells were difficult to identify and were in small numbers compared with other neural-derived cells, such as astrocytes and neurons.4–6 Since JCV does not infect neurons in the brain or in cultures, astrocytes were implicated as the predominant cell type in these cultures in which JCV multiplied.7 This observation was later confirmed both in situ in histopathological samples and in cell cultures with the establishment of astrocyte cell lines susceptible to JCV infection.8,9 Although cultures from fetal brain tissue demonstrate a diverse population of cell types, it has been possible to prepare cultures highly enriched for astrocytes. It has been these astrocyte-pure cultures from human fetal brain that have served as cell substrates for studies of neurotropism of viral pathogens. With the observation that HIV-1 was neuroinvasive (a characteristic of the lentivirus family), and was demonstrated in patients with dementia, laboratory and clinical investigations began to determine whether HIV-1 was also neurotropic, i.e., able to infect neural-derived cells. Astrocyte cell cultures from human fetal brain were used to initiate these studies.

Electrochemiluminescence-Based Detection System for the Quantitative Measurement of Antigens and Nucleic Acids: Application to HIV-1 and JC Viruses

A system for quantitative measurements based on electrochemiluminescence (ECL) has recently been developed. ECL is the generation of light through a series of chemical reactions at an electrode surface using a label that is a chelate of ruthenium(II) tris(2,2′-bipyridine) (Rubpy). ECL is ideally suited for analytic procedures involving antigens or nucleic acids because of the precision, sensitivity, and accuracy of the system. In addition, assay formats which eliminate wash steps and have rapid kinetics have been developed.