The experiments in this application are designed to elucidate the biology of an oligodendrocyte-specific virus, JC virus. JC virus causes a fatal demyelinating disease (progressive multifocal leukoencephalopathy) of immunosuppressed patients. Glial-specificity is controlled by the viral promoter. We isolated JC viral promoters directly from the brains of patients with PML. Each viral promoter contains an identical proximal sequence (including an Spl binding site that we have recently characterized) that is highly similar to myelin basic protein and proteolipid protein promoters, but dissimilar to the original, culture derived JC virus isolate (Mad-1 strain). We hypothesize that this proximal, conserved promoter region contributes to the glial-specificity of JC virus gene transcription Our aims are to: 1) examine the transcriptional function and specificity of the conserved JC virus promoter region using promoter-CAT constructs in transfection assays, 2) to test the function and specificity of the Spl binding site in the proximal JC virus promoter, using mutant promoters in CAT assays and Spl overexpression, 3) to test the ability of the chemotherapy agent mithramycin to inhibit Spl from binding to the JC virus promoter and to specifically inhibit JC virus transcription (thus becoming a potential therapeutic agent for PML), 4) to test JC virus promoter activity following stable chromosomal integration using retroviral vectors, 5) to isolate glial proteins that bind to the Spl site. In addition we plan to examine JC virus promoters isolated from healthy kidney to further identify sequences important for tissue specificity. The results of these studies have implications for the treatment of PML, for understanding glial-specific transcription, and for the design of brain-specific gene therapy.
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