Transcriptional regulation of human neurotropic papovavirus, JCV

Kumar, Kotlo Umesh (1994) Transcriptional regulation of human neurotropic papovavirus, JCV. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The human papovavirus JC virus (JCV) replicates only in glial cells and exhibits strict cell-specificity for expression. JC virus (JCV) has been frequently found in the plaques of brains of victims of the fatal demyelinating brain disease, progressive multifocal leukoencephalopathy (PML). PML is associated with immunodeficiency and has been identified recently as a frequent complication of acquired immunodeficiency syndrome (AIDS) . The regulatory region of JCV is contained mainly within the two 98 base pairs (bp) tandem repeats and is functional in both directions, with the early and late promoter-enhancer (JCVE and JCVL) controlling expression of large and small tumour antigens and viral structural proteins, respectively. -- Previously, it was shown in our laboratory that the regulatory region of JCV confers glial cell-specificity to JCV and contains three potential nuclear factor 1 (NF1) motifs (Nakshatri et al. 1990a). One motif towards the late side of the genome has a 3 bp palindromic repeat and the other two located within the 98 bp repeats have 6 bp inverted palindromic sequences. -- My initial objective was to evaluate the functional role of NF1 motifs in the restricted cell-specificity of JCV. This was approached by site-directed mutagenesis of the nuclear factor 1 (NF1) motifs within the viral regulatory region. The NF1 motifs, located within the 98 bp tandem repeats which contain 6 bp perfect inverted palindromic sequences were important for glial cell-specific expression of JC virus in differentiated embryonal carcinoma cells in vivo. The NF1 site on the late side of the repeats was not important, an observation confirmed by in vitro transcription studies. These observations were correlated with in vitro DNase I footprinting and mobility shift assays, which demonstrated specific interactions of factors in glial cell nuclear extracts with NF1 sites. -- To characterize the brain-specific factor(s) inducing the expression of JCV, a cDNA encoding the factor binding to NF1 II/III located in the repeats was isolated by screening the cDNA library prepared from retinoic acid-differentiated P19 embryonal carcinoma cells by Southwestern blotting. Co- transfection of cDNA with JCVE and JCVL reporter plasmids resulted in the expression of JCV in nonglial cells such as HeLa cells. These results suggested that NF1 II/III plays a key role in the glial cell type-specificity of JCV. -- Mutations in all the three potential NF1 binding sites greatly reduced the transcriptional activity of JCV early promoter-enhancer in glial cells. However, a residual greater than basal level of activity was still observed. Examination of the JCV promoter-enhancer sequences revealed a cyclic AMP response element (CRE) motif, TGAGCTCA, 4 bp from the NF1 II/III binding site in the repeats. This increased the transcriptional activity of JCVE after treatment with the cAMP analogue dibutyryl cAMP and cyclic AMP inducer forskolin. Mutations in CRE motifs abolished the induction of JCV expression by cAMP. In vitro binding studies showed the interaction of an approximately 43 kDa protein with CRE oligonucleotide. The results indicated an additive effect of NF1 II/III and CRE in the glial cell-specific expression of JCV. -- JCV large T-antigen functionally transactivates the viral late promoter only in glial cells and the molecular mechanism for transactivation has remained elusive. The NF1 sequences of JCV and the amino acids of JCV T-antigen required for the transactivation have remained unclear. I performed experiments to address these issues. Using site-directed mutagenesis of NF1 sites of JCVL, the integrity of nuclear factor 1 (NF1) binding motifs in the 98 bp repeats (NF1 II/III), but not the motif external to the repeats, was found to be essential for the induction of JCVL activity by JCV T- antigen, a result which was confirmed by in vitro transcription assays. Mobility shift assays detected the increased binding of NF1 to the NF1 II/III sequences in the presence of T-antigen. This suggests that JCV T-antigen facilitates the increased binding of NF1 to NF1 II/III site, thereby activating the late promoter of JCV. A model describing the mechanism has been proposed. -- The region(s) of T-antigen necessary for the transactivation of JCVL was assessed with a series of constructs containing mutated coding sequences which I generated. The amino acids 196-437 region of JCV T-antigen was required for most of the transactivation of JCVL and transformation of primary baby rat kidney cells.

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