Evidence for a mechanism of demyelination by human JC virus: Negative transcriptional regulation of RNA and protein levels from myelin basic protein gene by large tumor antigen in human glioblastoma cells

Abstract

Human JC virus (JCV) is a neurotropic human polyomavirus that was found in the plaques and oligodendroglial cells of the brains of patients with the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Transgenic mice expressing JCV large tumor (T)-antigen from integrated DNA showed dysmyelination in the central nervous system. However, the role of T-antigen from episomal DNA in the demyelination in PML remains unclear. In this report, we examined the effect of episomally expressed JCV T-antigen on the expression of myelin basic protein (MBP) in U-87 MG human glioblastoma cells to study the mechanism of demyelination. Expression assays of the MBP promoter in U-87 MG detected a 2.5-fold reduction in cells expressing intact T-antigen. Next, U-87 MG expressing T-antigen were examined by RNase protection assays for mRNA accumulation from the endogenous MBP promoter. Also, the expression of the MBP promoter plasmid was determined using in vitro transcription assays with extracts from T-antigen expressing cells. Both assays found a similar down-regulation of the MBP promoter by T-antigen, confirming that negative regulation occurred at the transcriptional level for the endogenous and exogenous MBP promoters. Furthermore, in situ immunofluorescence assays and quantitative Western blot analysis provided convincing evidence of a similar reduction in the level of MBP produced from the functional endogenous gene in U-87 MG glioblastoma cells expressing T-antigen. Thus, we provide evidence for the role of T-antigen in a transcriptional control mechanism for the demyelination that is caused by JCV in PML patients.

Identification of Key Genes and Signaling Pathways Associated with the Progression of Glioblastoma multiform

“…Similarly, GO enrichment analysis for down regulated genes was performed. Enriched genes such as MAG (myelin associated glycoprotein) [176], ASIC2 [177], MBP (myelin basic protein) [178], CNP (2’,3’-cyclic nucleotide 3’ phosphodiesterase) [179], CPEB3 [180], SLC8A2 [181], PRKCZ (protein kinase C zeta) [182], RELN (reelin) [183], CYP46A1 [184], SNAP91 [185], CNTN2 [186], NPY (neuropeptide Y) [187], RGS4 [188], IL1RAPL1 [189], ERBB3 [190], SH3GL2 [191], SH3GL3 [192], ARRB1 [193], DNM3 [194], SPOCK1 [195], CCK (cholecystokinin) [196] and INA (internexin neuronal intermediate filament protein alpha) [197] were identified with progression of GBM. Decrease expression of enriched genes such as such as SCN8A [198], BRSK1 [199], ANKS1B [200], CALB2 [201], GRM3 [202], BCAS1 [203] and CLCA4 [204] were responsible for advancement of different cancer types, but low expression of these genes were identified first time in GBM and may be involved in progression of GBM.…”

Olf-1, a Neuron-specific Transcription Factor, Can Activate the Herpes Simplex Virus Type 1-Infected Cell Protein 0 Promoter

“…All cell lines were maintained in Earle's minimum essential medium supplemented with 10% fetal bovine serum. Transfections were performed in 100-mm plates with the indicated amounts of DNA by calcium phosphate precipitation as described previously (24).…”

“…The amount of DNA transfected was kept constant in all experiments with pUC19. Cells were harvested 48 h after transfection and CAT enzymatic levels measured as described previously (24). The amount of extract used for measuring CAT was adjusted based on ␤-galactosidase activity as described previously (25).…”