BAG-1, a novel Bcl-2-interacting protein, activates expression of human JC virus

Laxminarayana R. Devireddy1, Kotlo U. Kumarc,1, Mary M. Paterb,2 and Alan Pater1

Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St John’s, Newfoundland, Canada1

Author for correspondence: Laxminarayana R. Devireddy. Present address: Howard Hughes Medical Institute, University of Massachusetts Medical Center, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA. Fax +1 508 856 5473. e-mail Laxminarayana.Devireddy{at}umassmed.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCVE promoter and to a lesser extent the JCVL promoter. Mutations in the NF-1 sites in the JCVE promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
The human polyomavirus JC virus (JCV) is the aetiological agent of progressive multifocal leukoencephalopathy (PML) (reviewed in Major et al., 1992 ; Weber & Major, 1997 ). This virus is closely related to two other polyomaviruses, the simian virus 40 (SV40) and BK viruses, which share more than 70% identity in their protein-coding regions (Frisque et al., 1984 ). However, compared with BK virus and SV40, JCV has a narrower host range and tissue tropism. The greatest divergence between JCV and the other polyomaviruses is in the virus control region, which contains promoters for early and late gene transcription and is organized as two 98 bp direct repeats. Several lines of evidence gained in vitro and in vivo, including in vitro transcription from the virus promoters in neuronal and non-neuronal cell extracts (Ahmed et al., 1990 ; Kumar et al., 1993 ) and transient transfection of glial and non-glial cells with CAT reporter plasmids (reviewed in Raj & Khalili, 1995 ), indicated that the JCV early and late promoters are more active in cells derived from the central nervous system (reviewed in Raj & Khalili, 1995 ). Thus, these data suggest that the tissue tropism of JCV, at least to a large extent, rests on tissue-specific expression of the virus promoters. Previous studies have indicated that the cell-specific activation of the JCV promoter requires its association with multiple cellular transcription factors present in neuronal cells (reviewed in Raj & Khalili, 1995 ).

Embryonal carcinoma (EC) cells are pluripotent in nature and can be differentiated with chemicals into several different cell lineages. For example, retinoic acid (RA) and DMSO induce neuron- and skeletal muscle cell-like morphologies, respectively. Thus, the EC cell system serves as a model system for studying neuron-specific gene expression. Previous studies from our laboratory have shown that the JCV promoter is active only in RA-differentiated and not in undifferentiated or DMSO-differentiated EC cells (Nakshatri et al., 1990 ; Kumar et al., 1993 ). Nuclear extracts prepared from RA-differentiated cells protect nuclear factor-1 (NF-1)-like sequences in the JCV promoter (Nakshatri et al., 1990 ; Kumar et al., 1993 ) and disruption of these sites reduces the activity of the JCV promoter (Kumar et al., 1993 ). Furthermore, a novel factor present in RA-differentiated EC cells seems to interact with these sites, as judged by the results of band-shift and UV cross-linking assays (Kumar, 1994 ). However, the identity and nature of this protein remain unknown. Cloning and characterization of such factors may shed more light on neuron-specific expression of human JCV.

BAG-1 (Bcl-2-associated athano gene-1) is a novel Bcl-2-interacting protein that was first identified as a multifunctional molecule that binds to the anti-apoptotic protein Bcl-2 and promotes cell survival (Takayama et al., 1995 ). Since its discovery, BAG-1 has been shown to form complexes with several other proteins, including tyrosine kinase growth-factor receptors such as hepatocyte- and platelet-derived growth factors (Bardelli et al., 1996 ), the serine/threonine protein kinase Raf-1 (Wang et al., 1996 ; reviewed in Wang & Reed, 1998 ) and the retinoic acid receptor (Liu et al., 1998 ). Alternative translation of BAG-1 generates a series of proteins with variable lengths (Packham et al., 1997 ). Longer forms of BAG-1, with unique amino-terminal domains, have also been reported to form complexes with steroid-hormone receptors (Packham et al., 1997 ; Froesch et al., 1998 ). Binding of BAG-1 to these molecules modulates their activity. The mechanism by which BAG-1 influences the activities of such diverse proteins can perhaps be attributed to its ability to bind heat-shock proteins directly, which in turn interact with multiple target proteins in cells. BAG-1 contains an amino-terminal region with similarity to ubiquitin and a central region that binds Bcl-2 (Takayama et al., 1995 ). Its carboxy terminus is required for formation of complexes with Raf-1 and growth factor receptors.

It is well established that the expression of JCV early and late genes, encoding the tumour (T) antigen and capsid proteins, respectively, determines the narrow host range and specificity of this virus. Studies from our lab and other laboratories have demonstrated that the JC promoter/enhancer functions more efficiently in neuronal cells than in non-neuronal cells. The studies presented here attempt to identify the proteins that bind the JCV promoter/enhancer region. Here, we report the cloning of a novel protein by Southwestern screening of a P19 RA-differentiated cDNA library with a JCV NF-1 probe. Interestingly, this protein is identical to a Bcl-2-interacting protein, BAG-1. Furthermore, this protein is able to bind the NF-1 sequence and transactivate JCV promoters.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells, plasmids and transfection procedures.
P19 EC, U87 MG human glioblastoma and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium. P19 cells were differentiated with 1% DMSO into skeletal or cardiac muscle cells or differentiated with 300 nM all trans-retinoic acid (Sigma) into neuron-like cells as described previously (Nakshatri et al., 1990 ). Plasmids pCMV, pBluescript KS(+) and pUC19 were purchased from Invitrogen, Stratagene and New England Biolabs, respectively. pJCVE, JCVL, RIIE, II10 (bearing mutations in two NF-1 sites present within the two 98 bp repeats) and DM10 (bearing mutations in all three NF-1 sites), CAT reporter plasmids and pRSV-{beta}-Gal were described by Nakshatri et al. (1990) and Kumar et al. (1993) . BKE CAT (BK virus early promoter linked to the CAT gene) was described by Nakshatri et al. (1991) . Cells were transfected with the amounts of plasmids indicated by the calcium phosphate method as described by Kumar et al. (1993) .

{blacksquare} Oligonucleotides.
The sequences of wild-type (WT) and mutant (mt) JCV NF-1 oligonucleotides used are: WT NF-1, 5' TGGCTGCCAGCCAA 3'; and mt NF-1, 5' gtaCTGCCAGaCcAGCA 3' (see Kumar et al., 1993 ). Lower-case letters indicate mutated bases.

{blacksquare} Construction and screening of the cDNA library.
A {lambda}gt22A cDNA library from mRNA of P19 RA-differentiated cells was made by using a kit from GIBCO-BRL according to the manufacturer’s instructions. Screening of the cDNA expression library was performed by the Southwestern blot method of Vinson et al. (1988) . Freshly prepared E. coli Y1090R- bacteria were infected with recombinant phage from the P19 RA {lambda}gt22A library at ~5x104 p.f.u. per 150 mm plate. The infection was performed by incubating at 37 °C for 30 min. Next, 7·5 ml 0·7% top agarose was mixed with the bacteria and the mixture was overlaid onto 1·5% T-Tyn (tryptone, yeast extract and NaCl) agar plates. After incubation at 42 °C for 4 h, IPTG-impregnated nitrocellulose filters were overlaid onto the plates and the plates were incubated for additional 6 h at 37 °C. The filters were then removed from the plates, air-dried and immersed in binding buffer (6 M guanidine–HCl; 25 mM HEPES, pH 7·9; 3 mM MgCl2; 4 mM KCl and 1 mM DTT) for 5 min at 4 °C with gentle agitation. The binding buffer was then diluted with an equal volume of the same buffer but containing variable amounts of guanidine–HCl and the filters were incubated in these diluted buffers for 5 min each. The filters were then washed with binding buffer without guanidine–HCl. The filters were then blocked by incubating in binding buffer containing 5% non-fat dried milk powder for 30 min at 4 °C. After rinsing in binding buffer containing 0·25% milk powder, the filters were probed with 106 c.p.m./ml nick-translated JCV NF-1 oligonucleotide concatemer in binding buffer containing 0·25% milk powder and 10 µg/ml sonicated salmon sperm DNA. After 3 h hybridization at 4 °C, the filters were washed three times for 15 min each with binding buffer containing 0·25% milk powder. Finally, the filters were wrapped in Saran Wrap and exposed to X-ray film overnight at -70 °C. Positive plaques were identified and re-screened until they were homogeneously positive.

{blacksquare} Sequence analysis.
The cDNA, which was cloned into pBluescript KS(+) (Stratagene), was sequenced by using a kit from United States Biochemical according to the manufacturer’s instructions. The sequence was compared with known sequences in GenBank by BLAST search.

{blacksquare} Northern blot analysis.
Total RNA from undifferentiated (UD), RA- and DMSO-differentiated EC cells, U87 MG and HeLa cells was extracted by using a kit from Promega, according to the manufacturer’s instructions. Twenty µg total RNA was electrophoresed on a 1% formaldehyde–agarose gel and transferred to nylon membrane (Gilman Sciences). The filter was blocked with Denhardt’s solution and probed with 106 c.p.m./ml nick-translated kNF-1 cDNA overnight at 60 °C. After hybridization, the filter was washed and exposed to X-ray film. The hybridized probe was stripped with 0·1xSSC and 0·1% SDS and reprobed with an actin probe as described above.

{blacksquare} Gel-shift analysis.
The sequences of JCV NF-1 oligonucleotides were described by Kumar et al. (1993) . The probes were prepared by end-labelling the oligonucleotides in the presence of [{alpha}-32P]dCTP. The binding reactions were done in a volume of 35 µl in buffer containing 10 mM Tris–HCl, pH 7·8; 1 mM EDTA; 5 mM DTT; 150 mM KCl; 12% glycerol; 10 µg poly(dI.dC), 10 ng labelled probe and 4 µl in vitro-translated BAG-1. The reaction mixture was incubated at room temperature for 30 min. The reaction products were resolved on a 4% non-denaturing PAGE gel. Gels were then dried and subjected to autoradiography.

{blacksquare} Southwestern blot analysis.
Nuclear extracts from UD, RA- and DMSO-differentiated EC cells, HeLa and U87 MG cells were resolved on a 10% SDS–PAGE gel and transferred to PVDF membrane in a semi-dry transfer apparatus (Bio-Rad) according to the manufacturer’s instructions. The filters were then blocked overnight at 4 °C with 5% milk powder in a binding buffer containing 25 mM HEPES–NaOH, pH 7·9; 5 mM MgCl2; 0·5 mM DTT and 25 mM NaCl. After blocking, the filters were probed with 106 c.p.m./ml nick-translated JCV NF-1 oligonucleotide in binding buffer. The filters were then washed with binding buffer without milk powder, dried and subjected to autoradiography.

{blacksquare} CAT assays.
CAT assays were performed as described previously (Nakshatri et al., 1990 ). The percentage of acetylation was quantified by liquid scintillation and normalized on the basis of {beta}-galactosidase activity.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Isolation of a cDNA encoding a JCV NF-1 region-binding protein from P19 RA-differentiated cells
Previous studies from our laboratory indicated that NF-1 sites present in the JCV enhancer play an important role in the neuron-specific expression of JCV (Nakshatri et al., 1990 ; Kumar et al., 1993 ). Furthermore, a novel protein seems to interact with the NF-1 motif (Kumar, 1994 ). This protein is present in both neuronal and non-neuronal cells and is able to interact specifically and directly with the JCV NF-1 motif. To examine the identity of this protein, an NF-1 oligonucleotide probe was used to screen a {lambda}gt22A cDNA expression library from RA-differentiated EC cells. After screening more than 2x105 plaques, two positive plaques were identified. The specificity of interaction of these plaques with the NF-1 probe was further confirmed. Replica filter papers containing plaques of the two positive recombinant phages were hybridized with: (i) WT NF-1; (ii) mt NF-1 (Kumar et al., 1993 ) and (iii) a non-specific oligonucleotide. Only the WT NF-1 probe (data not shown), and not the mutant or non-specific probes, interacted with the positive recombinant plaques (data not shown).

Analysis of DNA extracted from the two positive plaques yielded the same sequence, and only one plaque was characterized further. The positive recombinant phage encoding the JCV NF-1 region-binding protein was termed kNF-1 (Kumar, 1994 ). Analysis of kNF-1 revealed a ~1 kb DNA fragment (Fig. 1a) with perfect sequence identity to BAG-1 (Fig. 1b; Takayama et al., 1995 ). The kNF-1 cDNA contains an open reading frame (ORF) of 229 amino acids with the potential to encode a 30 kDa protein. The ORF corresponds to sequences spanning the entire BAG-1 sequence. BAG-1 is a Bcl-2-interacting protein isolated from a mouse embryonic cDNA library in the yeast two-hybrid system (Takayama et al., 1995 ). The sequence identity of kNF-1 to BAG-1 is likely to reflect the isolation of the same gene.



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Fig. 1. Sequence analysis of kNF-1 cDNA. (a) Nucleotide sequence of kNF-1 and the deduced amino acid sequence. Numbers on the left indicate the positions of nucleotides. Amino acid positions are indicated on the right. The first three start methionines are in bold. The polyadenylation signal is in bold and doubly underlined. (b) Amino acid sequence comparison of kNF-1 and BAG-1. The sequences were aligned by using the GAP program of GCG. Vertical lines indicate identity.

 
Northern blot analysis
JCV expresses its genes and replicates efficiently in brain cells (Kenney et al., 1984 ) and the NF-1 sites in the JCV regulatory region were shown to be critical for neuron-specific expression (Kumar et al., 1993 ). To determine the cell-specific expression of kNF-1, Northern blot analysis was performed with RNAs derived from UD, RA- and DMSO-differentiated EC cells, U87 MG and HeLa cells. Probing and washing were performed under high-stringency conditions to eliminate cross-hybridization. As shown in Fig. 2(a), a major ~1·5 kb RNA species was detected in UD, RA- and DMSO-differentiated EC cells, whereas a ~1·7 kb RNA species was detected in HeLa cells. From the intensity of the bands, it appears that kNF-1 RNA was present at approximately similar levels in all cell types with the exception of U87 MG cells, which did not express kNF-1 RNA. Stripping and reprobing the same blot with an actin probe showed that equal amounts of RNA were loaded on the gel (Fig. 2b). These observations indicate that a major 1·5 kb kNF-1 RNA species is produced in neuron-like cells.



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Fig. 2. Northern blot analysis of kNF-1 RNAs from various cell lines. Total RNAs were extracted from the cell lines as described in Methods and 20 µg RNA was used in Northern blot analysis. kNF-1 cDNA was used as a probe (a). The same blot was then stripped and reprobed with an actin probe (b). Numbers on the right indicate sizes of molecular mass markers (kb).

 
Nucleoprotein complex formation
The binding activities of crude nuclear extracts and in vitro-translated kNF-1 were tested by Southwestern and gel-shift analyses, respectively. To examine the ability of in vitro-translated kNF-1 protein to bind the JCV NF-1 oligonucleotide, a gel-shift assay was performed. As shown in Fig. 3(a), in vitro-translated kNF-1 protein reduced the mobility of the JCV NF-1 probe (lane 2). This retardation was eliminated by the addition of a 200-fold excess of unlabelled WT JCV NF-1 oligonucleotide (lane 3), but not by the addition of the mutant JCV NF-1 oligonucleotide (lane 4), confirming the specificity of binding of kNF-1 protein to the JCV NF-1 oligonucleotide. No retardation was observed with a reticulocyte lysate (lanes 5–7) or with in vitro-translated luciferase protein (lanes 8–10).



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Fig. 3. Analysis of DNA-binding properties of the kNF-1-encoded protein. (a) Gel-shift analysis of in vitro-translated kNF-1. Lanes: 1, free probe; 2–4, binding reactions containing 4 µl in vitro-translated kNF-1 protein with no competitor (lane 2), a 200-fold excess of WT JCV NF-1 oligonucleotide (lane 3) or a 200-fold excess of mutant JCV NF-1 oligonucleotide (lane 4); 5–7, binding reactions containing 4 µl rabbit reticulocyte lysate with no competitor (lane 5), a 200-fold excess of WT JCV NF-1 oligonucleotide (lane 6) or a 200-fold excess of mutant JCV NF-1 oligonucleotide (lane 7); 8–10, binding reactions containing 4 µl in vitro-translated luciferase protein with no competitor (lane 8), a 200-fold excess of WT JCV NF-1 oligonucleotide (lane 9) or a 200-fold excess of mutant JCV NF-1 oligonucleotide (lane 10). The arrow indicates the position of the free probe. (b) Southwestern blot analysis of nuclear extracts from indicated cell types. Nuclear extracts (50 µg) or BSA (50 µg) were resolved on a 10% SDS–PAGE gel, transferred to PVDF membrane and probed with WT JCV NF-1 oligonucleotide. The arrow indicates the position of the 30 kDa protein. The faster-migrating band in P19 RA-differentiated cells may be a proteolytically processed form. The numbers on the right indicate the positions of molecular mass markers (kDa).

 
The DNA-binding activity of kNF-1 was further confirmed in Southwestern (DNA–protein) blot assays. Nuclear extracts from the cell types indicated were resolved on an SDS–PAGE gel, transferred to PVDF membrane and probed with WT JCV NF-1 oligonucleotide. A 30 kDa protein was detected in P19 RA, P19 DMSO and HeLa cell extracts (Fig. 3b). The faster-migrating protein in P19 RA cells may be a proteolytically degraded kNF-1 (Devireddy, 1995 ). However, a 30 kDa protein was not detected in extracts from U87 MG glioblastoma cells. Furthermore, these cells are negative for kNF-1 mRNA expression (Fig. 2). A 30 kDa protein was also detected in extracts from P19 RA, P19 DMSO and HeLa cells in Western blot assays using a BAG-1-specific antibody (data not shown). The identity and nature of the 97 kDa protein present in all cell types are not known. However, a similar-sized protein present in glial and non-glial cells was also shown to interact with the JCV B domain (which harbours the NF-1 site) (Khalili et al., 1988 ). In summary, these results suggest that BAG-1 binds the JCV NF-1 oligonucleotide.

Transcriptional activity of kNF-1 cDNA in HeLa cells
To examine the transcriptional activity of the kNF-1 protein, the kNF-1 cDNA was cloned into a eukaryotic expression vector, pRc/CMV. Non-neuronal HeLa cells that do not support JCV transcription were employed (Tada et al., 1989 ). Reporter plasmids with the CAT gene under the control of the JCV early (JCVE) or late (JCVL) promoters were transfected into HeLa cells alone or with pCMV-kNF-1. A basal level of activity from the JCVE and JCVL promoters was detected in HeLa cells (Fig. 4a). Co-transfection of kNF-1 cDNA resulted in 6-fold and 5-fold activation of the JCVE and JCVL promoters, respectively (Fig. 4a). Co-transfection of pRc/CMV alone had no effect (data not shown). The RIIE expression plasmid, containing only one JCV 98 bp repeat, was also transactivated, by 2·5-fold (Fig. 4a). The BK virus early promoter was not transactivated by kNF-1 cDNA (Fig. 4a). A JCV enhancer/promoter containing mutations in the NF-1 sites was not transactivated by kNF-1, suggesting that the integrity of these sites is necessary for kNF-1-mediated transactivation (Fig. 4b). In summary, these results suggest that kNF-1 specifically transactivated JCV promoters.



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Fig. 4. Transactivation of JCV promoters by kNF-1. (a) Plasmids containing the CAT reporter gene under the control of the JCV early (JCVE) or late promoter (JCVL), the BKV early promoter (BKVE) and one 98 bp repeat (RIIE) were transfected (2 µg each) into HeLa cells alone or with 10 µg kNF-1 expression plasmid (pCMV-kNF-1). The graph below shows the adjusted percentage acetylation values. (b) Integrity of NF-1 sites is required for kNF-1-mediated transactivation. A JCV enhancer bearing mutations either in the two NF-1 sites present within the 98 bp repeats (II10 CAT) (lanes 1 and 2) or in all the three NF-1 sites (DM10 CAT) (lanes 3 and 4) (Kumar et al., 1993 ) was co-transfected with (lanes 2 and 4) or without (lanes 1 and 3) 10 µg kNF-1. Cell extracts were prepared 48 h post-transfection and CAT activity was measured. The graph below shows the adjusted percentage acetylation values.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In this study, we isolated a cDNA (kNF-1) encoding a JCV NF-1 motif-binding protein from P19 RA-differentiated cells that is identical to a Bcl-2-interacting protein, BAG-1 (Fig. 1). Furthermore, the kNF-1 protein bound specifically to the JCV NF-1 region and up-regulated transcription of the viral promoters in non-glial cells (Figs 3 and 4). Northern blot analysis for detection of kNF-1 mRNA revealed the presence of BAG-1 mRNA of varying sizes (Fig. 2).

JCV infection occurs during childhood in the majority of the population, but only during immunosuppression does this virus become pathogenic and cause PML. The virus replicates only in neuronal cells, but the mechanism that determines neuron-specific expression of virus genes is not completely understood. It has been postulated that a regulatory pathway that includes participation of neuronal and non-neuronal transcription factors plays a role in the neuron-specific regulation of JCV (reviewed in Raj & Khalili, 1995 ). The functional redundancy of promoter elements and the requirement for multiple cellular transcription factors further support the hypothesis of multiple cis and trans determinants of JCV neurotropism.

The data presented here led to the identification of a novel transcription factor, BAG-1, that bound specifically to the JCV NF-1 sequences and up-regulated transcription of the JCV early and late promoters. Recent studies indicate that BAG-1 and its longer isoforms BAG-1M and BAG-1L influence a wide variety of cellular phenotypes through their interaction with Hsc70/Hsp70, including resistance to apoptosis, promotion of cell proliferation, enhancement of tumour cell migration and alteration of transcriptional activity of steroid-hormone receptors (Takayama et al., 1995 ; Wang et al., 1996 ; Bardelli et al., 1996 ; Liu et al., 1998 ; Froesch et al., 1998 ). The ability of the BAG-1 family of proteins to modulate the chaperone activity of Hsc70/Hsp70 may have different consequences for the various proteins whose functions are controlled by conformational change. For example, BAG-1 proteins have been reported to bind Bcl-2, Raf-1, androgen receptor and PDGF receptor and enhance their function. Conversely, interaction of BAG-1 with retinoic acid receptor, glucocorticoid receptor and Siah-1 seems to inhibit their activity (Takayama et al., 1995 ; Wang et al., 1996 ; Bardelli et al., 1996 ; Liu et al., 1998 ; Kullmann et al., 1998 ; Matsuzawa et al., 1998 ; Froesch et al., 1998 ). The Bcl-2 protein, which has an anti-apoptotic function, recruits cytosolic proteins to internal membranes, including Raf-1 and BAG-1 (Wang et al., 1996 ; Takayama et al., 1998 ). Bcl-2 also negatively regulates p53 transcriptional activity (Froesch et al., 1999 ), possibly by sequestering transcription factors necessary for p53 transcriptional activity. Analogously, BAG-1 may modulate JCV expression by interacting with transcription factors.

It is intriguing that a ubiquitously expressed, anti-apoptotic and chaperone regulator BAG-1 activates the expression of JCV. Many distinct promoter elements, including NF-1, contribute to the tropism of the JCV promoter (Nakshatri et al., 1990 ; Kumar et al., 1993 ). The restriction in expression of individual transcription factors to glial cells, including Tst-1, NF-1 and Sp1, may confer neuron-specific expression to JCV (Nakshatri et al., 1990 ; Henson et al., 1992 ; Wegner et al., 1993 ). Therefore, the relative abundance of transcription factors, rather than a single determining factor, may account for the neurotropism of JCV.


   Acknowledgments
 
This work was part of the MSc and PhD theses of L.R.D. and K.U.K., respectively, submitted to the Memorial University of Newfoundland. We thank Doreen Bailey and Jose Teodoro for help in preparation of the manuscript. This work was supported by grants from the National Cancer Institute of Canada (with funds from the Canadian Cancer Society) and the Medical Research Council of Canada. L.R.D. and K.U.K. contributed equally to this work.


   Footnotes
 
c Present address: Dept of Molecular Genetics, Univ. of Illinois–Chicago, 900 S. Ashland Avenue, Chicago, IL 60607, USA.

b This work is dedicated to the fond memory of Dr Mary M. Pater, who passed away on 2 November 1994.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 3 August 1999; accepted 21 October 1999.