The effects of AGGGAAGGGA pentanucleotide repeat on JC virus gene expression and the isolation and characterization of a cDNA encoding an AGGGAAGGGA binding protein

Liu, Mingfeng (1995) The effects of AGGGAAGGGA pentanucleotide repeat on JC virus gene expression and the isolation and characterization of a cDNA encoding an AGGGAAGGGA binding protein. Masters thesis, Memorial University of Newfoundland.

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Abstract

Human JC virus (JCV) is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disorder of the central nervous system. Although JCV is a widely-distributed polyomavirus and is oncogenic in animals, its strict dependence for viral growth and expression of JCV on human fetal glial cells has hindered detailed studies of the regulation of JCV early and late gene expression. Previous reports have shown that gene expression of JCV is regulated by viral T antigen and cellular transactivating factors, such as NF1. To date, several cellular factors regulating JCV early and late gene expression have been identified and isolated, and their binding site sequences or motifs in JCV DNA have been characterized. -- The AGGGAAGGGA pentanucleotide (penta) repeat motif of the JCV control region has been suggested to positively (stimulate) and negatively (inhibit) regulate JCV early and late gene expression. A similar sequence that negatively regulates the expression of c-myc was also identified in the upstream region of the c-myc gene. Several proteins ranging from 25 to 80 kDa have been identified which specifically interact with this penta repeat motif. Among them, a 56 kDa protein has been suggested to act as a repressor for the JCV late gene expression and a 70 to 80 kDa protein has been shown to be required for the early gene expression. Thus, there are several proteins interacting with this penta repeat motif and differentially regulating the JCV early and late gene expression. Therefore, it would be very helpful to isolate and characterize such penta repeat motif binding proteins. -- To study the regulation by the penta repeat motif for JCV expression, I cloned JCV promoter-enhancer fragments containing mutated penta repeat motif into a CAT reporter plasmid and transfected the plasmid into P19 cells differentiated by retinoic acid into glial cells and U87MG human glioblastoma cells. CAT assays showed that the pentanucleotide repeat motif within the JCV promoter-enhancer region upregulated JCV late expression and downregulated JCV early expression. Mobility shift assays revealed two protein complexes specifically bound to the wild type penta repeat motif in both glial and nonglial cell extracts. Two proteins of approximately 60 and 45 kDa were detected by UV crosslinking. The penta repeat motif was then used to isolate a penta repeat motif binding factor by screening a cDNA expression library from retinoic acid-differentiated P19 glial cells. One positive cDNA clone (RP1) was found to specifically bind to the penta repeat motif. To characterize RP1, Southwestern blots with a β-galactosidase (β-gal) fusion protein indicated that this RP1 cDNA encoded the protein that specifically binds to the penta sequence. Consistent with my mutation results, preliminary in vivo results showed that the protein encoded by this cDNA could negatively regulate JCV early gene expression. The sequence of RP1 cDNA was found to contain sequences that were predicted to code for a protein containing seven putative zinc finger motifs in the carboxyl terminus two-thirds of the protein. Based on my data and the results of others, I proposed a model for the interaction of RP1 with JCV penta repeat motif as a complex in glial and nonglial cells, suggesting that this interaction contributes to the glial cell specificity of JCV. This model would involve RP1-penta acting in concert with other sites and factors in the JCV promoter-enhancer to prevent JCV gene expression in nonglial cells.

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