Characterization of a cDNA encoding a novel transcription factor regulating expression of human neurotropic JC virus

Devireddy, Laxminarayana R. (1996) Characterization of a cDNA encoding a novel transcription factor regulating expression of human neurotropic JC virus. Masters thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (26MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

JC virus (JCV) is a ubiquitous human neurotropic polyomavirus and is the etiological agent of the human demyelinating disease, progressive multifocal leukoencephalopathy (PML). Clinically, PML is found when immunodeficiency is caused by neoplasia,chronic disease, the immunosuppressive chemotherapy used for organ transplantation and, more recently, AIDS. JCV is closely related to two other polyomaviruses, BKV and SV40. Unlike BKV and SV40, JCV has a narrow tissue-tropism to glial cells. Several lines of evidence were used to attribute the JCV neurotropism to the viral regulatory region, which is organized as two 98 bp repeats. -- Previous studies from our laboratory demonstrated glial cell-specific expression of JCV in differentiating P19 embryonal carcinoma cells. The specificity was shown to be conferred by the NF-1 motifs present in the JCV regulatory region (Nakshatri et al., 1990a; Kumar et al., 1993). A cDNA encoding a factor that binds to the JCV NF-1 motif was cloned from a P19 glial cell cDNA library (Kumar, 1994). -- The objective for this thesis was to characterize this cDNA to understand its role in the expression and lifecycle of JCV. First, the cDNA sequence was determined. The analysis of the sequence of the cDNA revealed a significant homology both at protein and nucleic acid levels to a recently characterized factor interacting with Bc1-2 proto-oncogene protein called BAG-1. However, the cDNA isolated in our laboratory has a different 3'-end (nt 710-973). Therefore the cDNA was named bag-2. The 3'-end of the cDNA appeared to be alternatively spliced, because the point of divergence of the cDNA sequence from that of bag-1 was a consensus splice site. -- The bag-2 cDNA encoded a major 30 kDa protein, as determined by in vitro translation. The in vitro-translated BAG-2 protein was bound specifically to the JCV NF-1 II/III oligonucleotide and to the JCV enhancer, as determined by mobility shift and Southwestern blot assays. Further, the transfected bag-2 cDNA specifically transactivated JCV early (JCVE) and late (JCVL) promoters in non-glial HeLa cells and the transactivation required the integrity of the JCV NF-1 motifs. The recombinant BAG-2 produced in bacteria stimulated JCVE transcription in vitro by 3-fold, further suggesting that BAG-2 controls transcription. Interestingly, overexpression of BAG-2 in P19 glial cells inhibited the activated, but not the basal, level of transcription of both JCVE and JCVL in a dose dependent manner. However, such an inhibitory effect was not observed in U87 MG human glioblastoma cells. These results are reminiscent of the squelching observed for eukaryotic transcription factors. The bag-1 and 2 mRNAs were expressed in mouse embryonal carcinoma and human cervical cells but not in U87 MG cells. However, the BAG-2 specific C-terminus was expressed only in P19 cells. -- Deletion analysis of BAG-2 revealed that the C-terminus (a.a 195-229) was essential for transactivation, while the central α-helical region was important for DNA-binding. Though, the BAG-2 C-terminus was observed in P19 UD, glial and muscle cells, only in P19 glial cells was BAG-2 translocated into the nucleus. This was further confirmed by Southwestern blot analysis with nuclear extracts from these cells. -- The BAG-1 is important for modulating cell death. Since BAG-2 showed a significant homology to BAG-1, I tested the effect of BAG-2 on p53 promoter to delineate its possible role in apoptosis. The BAG-2 negatively regulated the mouse p53 promoter, as revealed by CAT assays. Thus, the BAG-2 might protect virus-infected cells from p53-induced apoptosis by decreasing the expression levels of p53. A model was proposed to explain the latency of JCV outside the central nervous system (CNS) and JCV glial cell-specific expression in CNS. In the model, BAG-2 is a novel transcription factor that modulates the expression of JCV differentially in glial and non-glial cells. In the former it activates the expression of JCV as a transcription factor in the nucleus, explaining the neurotropism of JCV. In the latter it inhibits the apoptosis induced by p53 thus, allowing the virus to persist for prolonged time.

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