Epigenetic regulation of polyomavirus JC.

BACKGROUND: Polyomavirus JC (JCV) causes the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), which occurs almost exclusively in people with immune deficiencies, such as HIV-1/AIDS patients. JCV infection is very common and usually occurs early in life. After primary infection, virus is controlled by the immune system but, rarely when immune function is impaired, it can re-emerge and multiply in the astrocytes and oligodendrocytes in the brain and cause PML. Thus a central question in PML pathogenesis is the nature of the molecular mechanisms maintaining JCV in a latent state and then allowing reactivation. METHODS: Since transcription can be regulated by epigenetic mechanisms including DNA methylation and histone acetylation, we investigated their role in JCV regulation by employing inhibitors of epigenetic events. RESULTS: The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate powerfully stimulated JCV early and late transcription while the DNA methylation inhibitor 5-azacytidine had no effect. Analysis of JCV mutants showed that this effect was mediated by the KB element of the JCV control region, which binds transcription factors NF-κB p65, NFAT4 and C/EBPß and mediates stimulation by TNF-α. Stimulation of transcription by p65 was additive with TSA as was cotransfection with transcriptional coactivators/acetyltransferase p300 whereas depletion of endogenous p65 by RNA interference inhibited the effect of TSA. EMSA with a KB oligonucleotide showed p65 expression, TNF-α stimulation or TSA treatment each caused a gel shift that was further shifted by antibody to p65. CONCLUSIONS: We conclude that JCV is regulated epigenetically by protein acetylation events and that these involve the NF-κB p65 binding site in the JCV control region.

Lentiviral transduction of neuronal cells.

Here we describe a general method for the construction of a lentivirus vector using a specific example of the construction of a lentivirus containing the luciferase reporter gene under the control of two hypothetical promoters and derived HIV-1 based lentivirus expression vector pLVX-Puro. This method can be used to compare the strength and regulation of different promoters. In this example, the target cells for transduction are human primary fetal astrocytes but the method is applicable to any primary cell culture from the CNS or other tissue and can be used to examine the strength of a particular promoter in different cell types. HIV based lentivirus particles are prepared by transfection of 4 plasmids into 293T cells using the Fugene 6 transfection reagent.