©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell-specific Activation of the Glial-specific JC Virus Early Promoter by Large T Antigen (*)

John W. Henson (§) , Bernd L. Schnitker , Tien-Shun Lee , Jon McAllister

From the (1) Molecular Neuro-Oncology Laboratory, Massachusetts General Hospital-East, Charlestown, Massachusetts 02129

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

JC virus causes the human demyelinating disease progressive multifocal leukoencephalopathy by selective infection of glial cells. This cell specificity results from glial-specific expression of viral early genes (large and small T antigens). Analysis of transcriptional regulation by the MH1 JC virus early promoter demonstrates that glial specificity is directed by the basal promoter. Because T antigen regulates the basal region of several viral and cellular promoters, we investigated whether it controls the JC virus basal promoter in a glial-specific manner. A JC virus T antigen expression plasmid generated a 95-kDa protein which exhibited nuclear localization and physical association with p53. T antigen repressed the JC virus and SV40 early promoters 4- to 5-fold in glioma cells. Conversely, T antigen induced 100- to 200-fold activation of the JC virus early promoter in nonglial cells, whereas the SV40 promoter was repressed. Activation required the JC virus TATA box sequence and a pentanucleotide repeat immediately upstream of the TATA box, but was independent of the upstream enhancer region. These data demonstrate that the JC virus basal promoter is responsible for glial-specific gene expression and suggest a mechanism for this regulation.


INTRODUCTION

JC virus causes a fatal, AIDS() -associated demyelinating brain disease (progressive multifocal leukoencephalopathy or PML) by infection of oligodendrocytes. Oligodendrocytes are the only cells in the body known to be lytically infected by JC virus, but viral early genes (i.e. large and small T antigen) are also expressed in astrocytes, which take on a bizarre, transformed appearance (1) . Attempts to isolate JC virus from PML brain met with failure until it was discovered that human fetal glial cells supported lytic infection in cell culture, further suggesting the glial-specific nature of the viral infection (2) . Molecular analysis revealed JC virus to be a 5.1-kb double-stranded, circular DNA virus in the Papovaviridae family with a remarkable degree of homology to SV40 (3) . As with SV40, JC virus genes are physically divided into two transcriptional units: those genes expressed early and late after infection. A single, bidirectional transcriptional regulatory region lies between these 2 units.

The JC virus early promoter directs higher levels of expression of a reporter gene in glial cells compared to nonglial cells (4, 5) . The DNA sequence of the JC virus promoter region varies considerably between isolates starting immediately upstream of the viral basal promoter region, which is highly conserved (6) . The MH1 form of the JC virus promoter contains a single basal promoter region, compared to the duplicated TATA regions in the original Mad-1 JC virus isolate, and has facilitated functional dissection of the elements involved in transcriptional regulation (Fig. 1). In a transient transfection analysis, the MH-1 JC virus early promoter activated 30- to 40-fold more transcription in glial cells than in nonglial cells. Using a sensitive luciferase assay, we found that upstream promoter elements activated expression in both glial and nonglial cells (5) . These results implicated the JC virus basal promoter in the regulation of glial specificity, and, therefore, we undertook a functional analysis of the basal promoter region.


Figure 1: Comparison of MH1 and Mad-1 JC virus early promoters. A, schematic of promoter structure. Open boxes represent tandemly repeated sequences that comprise the enhancer regions, the striped box in the MH1 promoter is the GA box (Sp1 binding site), the black box is the TATA box sequence, and the arrow marks the transcription initiation site. Shaded boxes represent sequences with homology to the large T antigen binding sites in the SV40 promoter. Note that the Mad-1 promoter has duplicated TATA box regions. B, DNA sequence of basal promoter regions of MH1, Mad-1, and SV40. MH1 and Mad-1 diverge immediately upstream of a pentanucleotide repeat indicated by the bracket.




MATERIALS AND METHODS

Plasmid Construction, Transfections, and Expression Assays

JC virus promoter-reporter plasmids were constructed by inserting the promoter constructs described below into the luciferase expression plasmid pAPLUC (5) . The DNA sequence of all inserts were verified by dideoxy sequencing (U. S. Biochemical Corp.). Full-length JC virus promoter (pMH1luc, Fig. 7, promoter B) was constructed using the MH-1 JC virus promoter (promoter p2, (6) ) and primers (JC6 5`-AGAAGCTTCACGTGACAGCT, JC26 5`-AGACTGCAGTTAGCTTTTTGCAGCAA) to amplify a region that included T antigen binding site I and the two upstream tandem repeats. pMH1(-LTaI)luc, in which T antigen binding site I was deleted by virtue of the location of a HindIII site between T antigen sites I and II, was constructed by inserting a HindIII/MscI fragment of the MH-1 JC virus promoter into pAPLUC. pMH1(Sp1 mut)luc (Fig. 7, promoter C) contained mutations that abolish binding of Sp1 (5`-AGCGAATACCT; sequence alteration is underlined). Deletion of the tandemly repeated enhancer sequences (pMH1(-TR)luc) was performed by amplification of the promoter fragment including the GA box and T antigen binding site I (primers JC26 and JC21 5`-TCCAGTTTTAGCCAGCTC; Fig. 7 , promoter D). pMH1(-Sp1)luc, which was identical with pMH1(-TR)luc except that the Sp1 site was also deleted, was similarly derived (primers JC26 and JC24 5`-ACCTTCCCTTTTTTTA; Fig. 7, promoter E). pMH1(TATA-SV)luc (Fig. 7, promoter F) and pMH1(TATA-irr)luc (Fig. 7, promoter G) contained the entire JC virus early promoter sequences of pMH1luc, but the TATA sequence was mutated either to that of SV40 (5`-TTTATTTATACA) or to an irrelevant sequence (5`-TTTAGCGTCACA) by PCR site-directed mutagenesis (7) . A pentanucleotide mutant pMH1(-P)luc contained irrelevant DNA sequence between the GA box and the polythymidine tract (5`-CCCTCCTCATGCAGGTTTTTTTT; Fig. 7, promoter H). pSVluc was constructed by ligating a HindIII/NaeI fragment of the SV40 control region, which contained all three T antigen binding sites, into pAPLUC. JC virus T antigen expression plasmid pJC-T was constructed by ligating a NotI-linked AciI/AvaI fragment of the early genomic region of the Mad-1 JC virus into pCR/CMV expression plasmid (Invitrogen). The orientation of the insert was confirmed by dideoxy DNA sequencing. SV40 T antigen expression plasmid (pSV-T; gift from Ulla Hansen, Dana-Farber Cancer Institute) contained the genomic early coding region of SV40 downstream of the RSV promoter. pCMV-p53 was a wild-type p53 expression vector (8) . Transient transfections were performed by a standard calcium phosphate method. 0.5 10 cells in 10-cm dishes were transfected with 20 µg of plasmid DNA (15 µg of promoter-luciferase plasmid, indicated amounts of T antigen expression plasmids, and pBSK (Stratagene) as a carrier). In the experiment in Fig. 1 , an SV40-CAT reporter plasmid was cotransfected to allow normalization between cell lines as described (5) . CAT activity was determined by a standard two-phase assay. Luciferase assays were performed in duplicate as described (5) . Within T antigen expression experiments, luciferase values were normalized by protein concentrations as determined by a bicinchoninic acid assay (Pierce), since T antigen regulates the promoters commonly used for internal controls (e.g. RSV promoter). All transfection assays were performed at least three times.


Figure 7: Regulation of JC virus basal promoter by T antigen. Transfections were performed using 0 or 4 µg of pJC-T/plate, the cells were harvested 48 h after transfection, and luciferase expression was measured and normalized to protein concentration. Luciferase expression in the presence of T antigen was divided by expression in the absence of T antigen, thus negative values represent -fold repression of the JC virus promoter, whereas positive values represent -fold activation. Promoter A is the wild-type Mad-1 promoter, B is wild-type MH1 promoter, C contains mutations in the Sp1 binding site, D lacks the upstream repeats, E lacks the Sp1 binding site and upstream repeats, F and G contain mutations in the TATA sequence to that of SV40 (F) and an irrelevant sequence (G), and H has mutations across the pentanucleotide sequence shown in Fig. 1.



Immunocytochemistry, Immunoprecipitation, and Western Analysis

U87MG glioma cells were plated in 6-well plates and transfected with either pJC-T or pBSK. After 48 h, the cells were fixed in ethanol for 10 min, treated with 0.3% HO for 10 min, blocked in 10% horse serum in PBS for 10 min, and incubated with 10 µg/ml anti-large T antigen antibodies (PAb 416, Oncogene Science) in 2% BSA/PBS for 1 h at room temperature. Following PBS washes, cells were incubated in a 1:200 dilution of biotinylated horse anti-mouse secondary (Vector) in 2% BSA/PBS at room temperature for 1 h, washed with PBS, incubated in avidin-biotin complex (Vector) for 1 h at room temperature, and the chromogen was developed with diaminobenzidine (Sigma). For Western analysis of T antigen, equivalent amounts of 1% Nonidet P-40 or 1% Triton X-100 lysate protein were denatured in SDS buffer and separated by 8% SDS-PAGE. Following transfer to nitrocellulose, T antigen was identified using 15 µg/ml primary antibody in 2% BSA/TBST (PAb 416 anti-large T antigen antibody, Oncogene Science) and peroxidase-labeled horse anti-mouse secondary at a dilution of 1:7,500 in 2% BSA/TBST. The peroxidase label was detected by enhanced chemiluminesence (ECL, Amersham). Immunoprecipitations were performed with 1% Nonidet P-40 cell lysates using an agarose-conjugated anti-large T antigen antibody (PAb 416, Oncogene Science). Following denaturation in SDS loading buffer, the immunoprecipitated complexes were separated by 10% SDS-PAGE and transferred to nitrocellulose (Schleicher and Schuell). Coimmunoprecipitation of p53 was detected by incubation of the filter with I-labeled anti-p53 antibodies (PAb 421, Oncogene Science), followed by autoradiography (Kodak).


RESULTS

The MH1 JC Virus Basal Promoter Directs Glial-specific Gene Expression

We compared the ability of JC virus early promoter mutants to activate gene expression in U87MG glioma cells and HeLa cervical carcinoma cells in a transient transfection assay. An SV40CAT reporter plasmid was cotransfected to allow for normalization of transfection efficiency and to allow a semiquantitative comparison of expression between the two cell lines (5) . Fig. 2shows the ratio of normalized luciferase expression between U87MG and HeLa cells for each promoter construct. Wild-type JC virus early promoter pMH1luc was 34-fold stronger in glial cells than in nonglial cells. A promoter with mutations in the GA box (Sp1 binding site) was 19-fold stronger in glial cells, and a deletion mutant which contained the Sp1 binding site but lacked the upstream repeats remained 19-fold stronger in glial cells. Together with our earlier observation that the upstream tandem repeats activated gene expression in both U87MG and HeLa cells (5), this demonstrated that the basal region of the JC virus early promoter directed glial-specific expression of a reporter gene.


Figure 2: The MH1 JC virus basal promoter directs glial-specific expression of a reporter gene. Ratios of normalized luciferase expression in U87MG glioma and HeLa cells are shown. Promoter A is the wild-type MH1 JC virus promoter, B contains mutations that abolish binding of Sp1, and C is the proximal promoter region including the Sp1 site.



Functional Analysis of the JC Virus Basal Promoter

We examined the function of the JC virus basal promoter by expressing a transcription factor that might regulate this region. The MH1 JC virus promoter has notable similarity to the SV40 promoter, which is strongly active in a wide variety of cell types and is repressed by its early gene product large T antigen (T antigen) through cooperative binding to three sites in the basal promoter (9) . T antigen activates the human hsp70 promoter in a manner that is dependent upon the exact sequence of the TATA box (10, 11) , and there is in vitro evidence that T antigen physically interacts with TATA binding protein (TBP) (12) . Based on these observations, it appears that T antigen regulates the basal promoters of viral and cellular genes during initiation complex formation. Because T antigen regulates various basal promoters in a divergent fashion, and because the TATA box sequences of JC virus and SV40 are different (Fig. 1), we used T antigen to investigate regulation of the glial-specific JC virus basal promoter region.

The early genomic region of Mad-1 JC virus was inserted into a cytomegalovirus expression plasmid (pJC-T), and the protein product generated by transient transfection of pJC-T in U87MG glioma cells was examined. Western analysis of cell lysates from pJC-T transfected cells demonstrated a 95-kDa protein (Fig. 3A, lane 3) that migrated slightly faster than the SV40 T antigen expressed from pSV-T (lane 2), consistent with the predicted number of amino acids (JC virus = 688 amino acids; SV40 = 695 amino acids) (3) , whereas mock-transfected cells contained no reactive species (lane 1). T antigen contained a functional nuclear localization signal, as shown by strong nuclear staining with anti-large T antibodies in transfected U87MG glioma cells (Fig. 3B), whereas mock-transfected cells exhibited no staining (Fig. 3C). Finally, we tested whether the pJC-T product could form a physical complex with p53. U87MG cells were cotransfected with an expression plasmid containing a human p53 cDNA and either pSV-T or pJC-T. Fig. 3D shows that p53 coimmunoprecipitated with the T antigens of both SV40 (lane 2) and JC virus (lane 3). No p53 was detected in cells transfected with p53 expression plasmid alone (lane 1). Thus, pJC-T directed expression of full-length JC virus large T antigen that was functional by criteria of nuclear localization and interaction with p53.


Figure 3: JC virus T antigen expression in U87MG glioma cells. A, Western analysis revealed a 95-kDa protein in U87MG cells transfected with pSV-T (lane 2) or pJC-T (lane 3). Mock-transfected cells (lane 1) showed no immunoreactivity. B, immunocytochemistry with anti-large T antigen antibody revealed strong nuclear staining in cells transfected with pJC-T. C, absence of staining in mock-transfected U87MG glioma cells (methyl green counterstain, 200). D, physical association between p53 and JC virus T antigen was demonstrated by immunoprecipitation with anti-large T antigen antibodies, followed by Western analysis with I-labeled anti-p53 antibodies. Lane 1 contained no transfected T antigen, lane 2 was a cotransfection of pSV-T and p53, and lane 3 was a cotransfection of pJC-T antigen and p53.



Regulation of the JC Virus Early Promoter by T Antigen

To determine the transcriptional effect of JC virus T antigen on the JC virus early promoter, pJC-T was cotransfected with JC virus promoter-reporter plasmids into U87MG glioma cells. SV40 T antigen is known to repress its own expression by binding to three T antigen binding sites in the SV40 early promoter (9) . The JC virus early promoter pMH1luc was repressed 5-fold by JC virus T antigen in a concentration-dependent manner (Fig. 4). Fig. 4also shows that JC T antigen, which has 72% amino acid identity to SV40 T antigen, repressed the SV40 promoter 5-fold. Repression was partially dependent upon the presence of binding site I, since deletion of site I in pMH1(-LTaI)luc resulted in weaker repression. Similar levels of repression were seen with transfections performed in another glioma cell line (T98G glioblastoma cells, data not shown).


Figure 4: Transcriptional repression of the JC virus (pMH1luc) and SV40 (pSVluc) early promoters by JC virus T antigen measured at 48 h after transfection into U87MG glioma cells. Repression was dependent upon binding site I, since a promoter in which this site has been deleted, pMH1(-LTaI)luc, was repressed less strongly than wild-type promoter.



Three-fold stimulation of the Rous sarcoma virus (RSV) promoter was seen in this assay, demonstrating that repression of the JC virus early promoter was not the result of a nonspecific reduction in gene expression. Analysis of the time course of transcriptional repression of the JC virus early promoter by T antigen (0.5 µg or 4 µg/plate at 0, 6, 12, 24, and 48 h following a 12-h transfection) revealed progressively stronger repression over time with maximal repression apparent between 24 and 48 h (data not shown). Although the promoter constructs contained the JC virus origin of replication, detectable plasmid DNA replication did not occur over the 48 h of the transient transfection assay as determined by Southern analysis of low molecular weight DNA extracted from the transfected cells (data not shown). Thus, transcriptional repression was not dependent on DNA replication. In conclusion, T antigen produced JC virus early promoter repression in U87MG glioma cells that was dependent upon concentration of T antigen and the presence of T antigen binding site I.

Comparison of JC Virus and SV40 T Antigens

The experiments in Fig. 4demonstrated that JC virus T antigen repressed both JC virus and SV40 early promoters. To compare the function of JC virus and SV40 T antigens, we tested their regulation of the SV40 promoter in U87MG glioma cells. Fig. 5A shows that both viral T antigens produced transcriptional repression of the SV40 early promoter, and that 50% repression occurred with lower amounts of transfected pJC-T than with pSV-T. The pMH1luc JC virus promoter was repressed by both T antigens in a similar manner (data not shown). Quantitative Western analysis also suggested that JC virus T antigen was a more potent repressor than SV40 T antigen (Fig. 5B); however, since the relative affinity of the antibody for the two T antigens is not known, this conclusion remains tentative. Thus, in glial cells, JC virus and SV40 early promoters and T antigens were functionally interchangeable.


Figure 5: Comparison of transcriptional repression of the SV40 early promoter (pSVluc) by JC virus and SV40 T antigens in U87MG glioma cells. A, titration of JC virus and SV40 T antigens revealed that both proteins repress the SV40 early promoter. 50% repression occurred with transfection of lower amounts of JC virus T antigen expression plasmid (pJC-T) compared to pSV-T. B, quantitative Western analysis of T antigen also suggested that JC virus T antigen was a more potent repressor.



Cell-type Specificity of T Antigen Promoter Regulation

We examined the possibility that repression was specific to glial cells. The SV40 promoter was repressed by T antigen in HeLa cells (Fig. 6), but T antigen produced 100- to 200-fold activation of the JC virus early promoter. A second nonglial cell line (JEG3 choriocarcinoma cells) gave similar results, although the level of activation was lower. SV40 T antigen also produced stimulation of the JC virus early promoter. There was no evidence of plasmid replication as determined by Southern analysis (data not shown). Activation was dependent upon a direct interaction between T antigen and the promoter, since pMH1(-LTaI)luc showed only slight stimulation (Fig. 6).


Figure 6: In HeLa cells the JC virus early promoter (pMH1luc) was stimulated strongly by T antigen, whereas the SV40 promoter (pSVluc) was repressed. Very weak activation was observed with the JC virus promoter lacking T antigen binding site I, pMH1(-LTaI)luc.



Promoter Elements Required for Transcriptional Activation

Having found that T antigen regulated the JC virus early promoter in a cell-specific manner, we sought to define promoter elements that were required for activation in HeLa cells. Activation was independent of the tandemly repeated enhancer region, since a reporter construct containing only the proximal promoter was still activated by T antigen (Fig. 7, promoters D and E). Recent data have suggested that T antigen activates the hsp70 promoter in a TATA sequence-dependent manner (10, 11) and that it interacts with TATA binding protein (TBP) (12), and TBP-associated repressors (e.g. Dr1) have been implicated in TATA sequence-dependent transcriptional regulation (13, 14) . We thus tested whether T antigen activation of the JC virus promoter was TATA-dependent. T antigen-induced repression was still seen in U87MG glioma cells following mutation of the JC virus TATA sequence to that of the SV40 promoter or to an irrelevant sequence (promoters F and G). In HeLa cells, however, T antigen failed to activate the pMH1(TATA-SV)luc promoter and the pMH1(TATA-irr)luc promoter, demonstrating that transactivation by T antigen was indeed dependent upon the sequence of a TATA box.

Comparison of the MH1 and Mad-1 basal promoter sequences revealed that the two promoters are identical up through a repeated pentanucleotide sequence immediately upstream of a polythymidine tract (Fig. 1B). A previous report suggested that the pentanucleotide sequence mediates repression of the early promoter in nonglial cells, based on relatively small changes in CAT activity (15) . To determine whether this sequence was required for T antigen-induced transactivation, the DNA sequence between the GA box and the polythymidine tract was replaced by an irrelevant sequence (Fig. 7, promoter H). T antigen was unable to stimulate a promoter containing the pentanucleotide mutation. However, this mutation did not increase the level of transcription in HeLa cells. Mutation of the GA box, which is immediately upstream of the pentanucleotide sequences, did not abolish the ability of T antigen to transactivate, demonstrating specificity for the pentanucleotide and TATA box sequences (Fig. 7, promoter C).


DISCUSSION

Once a rare condition, PML is no longer infrequent since approximately 5% of patients with AIDS develop this fatal brain disease (16) . The unique feature of PML is the selective destruction of oligodendrocytes by JC virus and selective expression of viral genes in astrocytes. Although it is likely that this cell-type specificity is regulated at more than one point in the viral life cycle, the best understood of these points is the glial-specific activation of early viral gene expression. Based upon features of transcriptional enhancers, it seemed likely that glial specificity would be mediated by the tandemly repeated elements. However, our data have demonstrated that the MH1 JC virus basal promoter region also has a role in this specificity.

We therefore examined the role of T antigen, which regulates the basal promoter, in JC virus early promoter regulation. We detected a divergent, cell-type-specific response to T antigen, and we found that these responses were mediated by sequences in the basal promoter. Whereas JC virus T antigen induced repression of the JC virus early promoter in glial cells, a different phenomena was observed in HeLa cells, where the JC virus early promoter was strongly stimulated.

Activation was dependent upon the sequence of the TATA box. The function of the TATA mutant JC virus promoter in glial cells in the absence of T antigen was not altered, demonstrating that the loss of activation in HeLa cells was not due to a nonspecific loss of promoter activity. Alterations in TATA sequences have been shown to exert specific effects on function of promoters. For instance, the sequence 5`-TATAA in the hsp70 promoter was crucial for activation by T antigen and the adenovirus protein E1A (11, 17, 18) . Replacement of this sequence with the SV40 TATA sequence 5`-TATTTAT abolished E1A-induced activation but maintained heat inducibility, whereas mutation to an irrelevant sequence resulted in loss of both E1A and heat inducibility. Upstream elements were not required for activation by T antigen. Tissue-specific expression from the myoglobin gene promoter required a specific TATA box sequence in concert with a muscle-specific enhancer element (19) . The recent identification of TATA-associated factors and the observation that T antigen binds to TBP suggest that specificity directed by the basal promoter may be achieved through the proteins that interact directly with TBP (20) .

Mutation of the pentanucleotide sequence adjacent to the TATA box also abolished activation by T antigen. The pentanucleotide sequence is of particular interest because it is the most upstream sequence shared by MH1 and Mad-1 promoters. Others have reported the presence of a possible repressor site in the JC virus Mad-1 promoter. Early gene expression in JC virus-transformed hamster glial cells was abolished following fusion of the glial cells with mouse fibroblasts, consistent with the presence of a protein in the nonglial cells that repressed the JC virus early promoter (21) . Further evidence for a specific repressor site in the JC virus early promoter came from studies in which an oligonucleotide, containing sequences which overlapped the viral TATA box, was introduced upstream of the SV40 basal promoter and was found to stimulate reporter gene expression in glial cells (U87MG glioma), but repress expression in mouse fibroblasts (15) . This analysis was performed using a heterologous promoter construct, and the changes in CAT activity were very small, making interpretation of the data difficult. Our pentanucleotide mutant did not exhibit increased transcriptional activity in HeLa cells, possibly because of the extent of the mutation.

Specific nuclear proteins bind to the pentanucleotide repeats in the basal promoter (22, 23) . These experiments have employed a combination of gel mobility shift assays and UV cross-linking of the protein to DNA to demonstrate the binding of an approximately 50-60-kDa protein present in glial and HeLa cells. The nature of this protein remains unknown.

Although there are several potential mechanisms for these results, they are most easily explained by the presence of a TBP-associated repressor that binds to the basal JC virus promoter and that is displaced by T antigen. A model for such a mechanism can be found with the adenoviral protein E1A, which appears to activate the hsp70 promoter in a TATA sequence specific manner by dissociating a 19-kDa repressor, Dr1, from TBP (14) . The hypothesis that the glial specificity of the JC virus early promoter is regulated by a repressor acting on the basal promoter in nonglial cells is attractive because it explains a number of experimental observations: it explains why the basal promoter is invariant, but the upstream enhancer areas vary between isolates (6) and why extensive searches have failed to reveal a clearly glial-specific element in the upstream promoter region. Finally, it would also explain our recent studies showing that the upstream enhancer regions activate the basal promoter in glial and nonglial cells (5) . Together with experiments demonstrating that the glial-specific transcription factor Oct-6 regulates the JC virus basal promoter (24) , these results suggest that the mechanism of glial specificity resides in basal elements instead of the enhancer region.


FOOTNOTES

*
This work was supported in part by NINDS Grant NS 01605 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Molecular Neuro-Oncology Laboratory, Massachusetts General Hospital-East, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-5510; Fax: 617-726-5079; E-mail: henson@helix.mgh.harvard.edu.

The abbreviations used are: AIDS, acquired immunodeficiency syndrome; CAT, chloramphenicol acetyltransferase; PML, progressive multifocal leukoencephalopathy; RSV, Rous sarcoma virus; TBP, TATA binding protein; PBS, phosphate-buffered saline; BSA, bovine serum albumin.


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