Functional Comparison of PML-Type and ... - Journal of Virology

JOURNAL OF VIROLOGY, Mar. 1996, p. 1512–1520 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 70, No. 3

Functional Comparison of PML-Type and Archetype Strains of JC Virus ELISABETH SOCK, KARIN RENNER, DANIELA FEIST, HUBERT LEGER, AND MICHAEL WEGNER* Zentrum fu ¨r Molekulare Neurobiologie, Universita ¨t Hamburg, D-20246 Hamburg, Germany Received 30 August 1995/Accepted 29 November 1995

Isolates of the human polyomavirus JC can be grouped as either PML-type or archetype strains primarily on the basis of divergence in their regulatory regions. Only PML-type viruses have so far been found to be associated with the human demyelinating disease progressive multifocal leukoencephalopathy. Here we have compared the functional properties of archetype and PML-type regulatory regions with regard to DNA replication and viral gene expression. No significant differences could be detected between archetype and PML-type regions in their ability to direct episomal DNA replication in the presence of JC virus T antigen. When viral gene expression was examined, early- and late-gene promoters from all PML-type strains exhibited a significantly higher activity in glial than in nonglial cells. Surprisingly, archetype strain promoters were also preferentially active in glial cells, although this effect was less pronounced than in PML-type strains. Furthermore, all promoters from archetype strains reacted to the presence of viral T antigen or the glial transcription factor Tst-1/Oct6 in a manner similar to the promoters of the PML-type viral strain Mad-1. Interestingly, T antigen and Tst-1/Oct6 were found to function in a species-specific and cell-type-specific manner, respectively. We conclude from our experiments that the differences in the regulatory regions cannot account for the different biology of archetype and PML-type viral strains. from PML (38, 58, 62, 63). These so-called archetype strains contain sequences in their regulatory regions not found in PML-type isolates. Most conspicuously, they lack the PMLtype tandem repeats. Because of their different association with PML, it has been hypothesized that the onset of PML might be functionally linked to a conversion of an archetype JC virus into a PML-type virus (5, 23, 32, 62, 63). It is intriguing to speculate that the glia specificity of viral gene expression is somehow attained during rearrangements within the viral regulatory region, making the conversion from archetype into PML type a necessary precondition for the observed tropism of JC virus. Similarly, it is conceivable that viral DNA replication could be altered by these rearrangements in a manner essential for productive lytic infection. The present study was undertaken to better understand the relation between archetype and PML-type strains. For this purpose, we compared the replicative behavior as well as the activity of early and late promoters from various JC virus isolates and analyzed their response to cellular and viral transactivators in various cell lines.

The human polyomavirus JC is very widespread within the human population (47). Infection usually results in a persistent virus that might be reactivated in patients who are in an immunosuppressed state. Under conditions of severe immunosuppression, JC virus can become an opportunistic pathogen, causing the demyelinating disease progressive multifocal leukoencephalopathy (PML) (for recent reviews, see references 16 and 35). Once a rare condition, PML has become one of the leading AIDS-associated neurological complications (6, 54, 59). During the course of PML, JC virus specifically infects glial cells and causes demyelination by destruction of myelinforming oligodendrocytes (35). Its tropism for glial cells in vivo is reflected by a highly restricted activity in cell culture, so that JC virus can be propagated only in primary human fetal glial cells or transformed lines derived from them (36, 37). Part of this tissue tropism can be attributed to preferential expression of viral genes in glial cells, which is reflected by a pronounced glial, specificity of transcription from both viral promoters (21, 25, 27, 29, 56). These promoters direct the expression of the early viral regulatory proteins as well as the late viral capsid components. They are both contained within the viral regulatory region, as is the origin of DNA replication (15). Despite substantial differences, regulatory regions of JC virus isolates from PML patients have a lot of features in common with the regulatory region of prototypic strain Mad-1, including the presence of large tandem repeats and the conservation of TATA elements and of binding sites for a number of transcription factors (5, 14, 32, 39, 61). Set apart from these PML-type strains is a second group of JC virus strains, which have been isolated most commonly from urine or kidney tissue of individuals who did not suffer from immunosuppression or

MATERIALS AND METHODS Plasmids. Cloned partial genomes for JC virus strains G2, N1, and MY were obtained from Y. Yogo (62), and GS/B-derived sequences were a gift of K. Do ¨rries (32). Full-length viral regulatory regions (spanning the region between the translational start codons for T antigen on the early side and for the agnogene on the late side) with XhoI restriction sites at their ends were generated by PCR with primer A (59-ACTTTCTCGAGTTTAGCTTTTTGCAGCAA-39 corresponding to map positions 5003-5031 of JC virus strain Mad-1) (15), and primer B (59-TCCCCCTCGAGCAGCTGGTGACAAGCCA, corresponding to map positions 284 to 257). For GS/B, two fragments were obtained because of alternative annealing of primer B within or at the end of the regulatory region. Only the longer fragment corresponding to the full-length regulatory region of GS/B was used in this study. Regulatory regions were cloned into pBKS-II1 (Stratagene), verified by dideoxy sequencing, and subsequently transferred into the luciferase reporter pGLbasic (Promega) in both possible orientations, thus yielding pGL-G2, pGL-N1, pGL-MY, pGL-GS/B, and pGL-Mad1. Depending on the orientation of the viral regulatory region in front of the luciferase gene,

* Corresponding author. Mailing address: ZMNH, Interim I, Pav. 22, Martinistr. 52, D-20246 Hamburg, Germany. Phone: 49 40 4717 4708. Fax: 49 40 4717 4774. 1512

VOL. 70, 1996

PROPERTIES OF PML-TYPE AND ARCHETYPE JC VIRUS STRAINS

early- and late-gene transcription could selectively be analyzed. Effector plasmids pRSV-JCT and pCMV-Tst-1 have been described previously (53, 60). Cell culture, transfections, luciferase assays, and replication assays. U138 cells were propagated in RPMI medium; U87, 33B, CV1, L tk2, and 293 cells were propagated in Dulbecco’s modified Eagle’s medium. All media were supplemented with 10% fetal calf serum. One day before transfection, cells were plated at a density of 5 3 105 cells per 60-mm plate in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Cells were transfected with 2 mg of luciferase reporter plasmid. For some experiments, 2 mg of expression plasmids for Tst-1 or JC virus T antigen were added. With the exception of 33B, cells were transfected by the calcium phosphate technique (8). To achieve maximal transfection efficiency, the precipitate was left overnight on CV1 and 293 cells. U138, U87-MG, and L tk2 cells were incubated with the precipitate for only 4 h before being treated for 1 min with 30% (vol/vol) glycerol in phosphatebuffered saline (PBS). 33B cells were transfected for 3 h with DEAE-dextran and exposed for 1 min to 10% (vol/vol) dimethyl sulfoxide in PBS. After transfection, cells were placed in fresh Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and harvested after 48 h for luciferase assays or after 72 h for replication assays. Luciferase assays and replication assays were performed as described previously (53, 60). Immunofluorescence. CV1 cells were seeded on chamber slides (Lab-Tec; Nunc Inc.) and transfected as described above. At 48 h posttransfection, the medium was removed and the cells were washed twice with PBS. The cells were then fixed with 3% formaldehyde in PBS for 20 min and treated with 1% Triton X-100 in PBS for 5 min. After being washed twice with PBS, the cells were incubated for 20 min with polyclonal anti-JCT antiserum (diluted 1:500; a gift of J. Gerber and F. Grummt) in PBS containing 0.1% Tween 20 (PBST) and 1% horse serum. After being washed three times with PBST, the cells were incubated for 20 min with Cy3-conjugated goat anti-rabbit antibodies (Dianova) diluted 1:500 in PBST. The cells were washed extensively with PBST, mounted, and analyzed on an Axiovert microscope (Zeiss). Preparation of nuclear extracts. Extracts were prepared as described previously (51). Briefly, cells from two 100-mm plates were washed twice with PBS, scraped from the plates in hypotonic buffer, swollen on ice, and lysed by the addition of 1% Nonidet P-40 and vortexing. Nuclei were pelleted and extracted for 15 min at 48C with constant rotation in 300 ml of ice-cold 10 mM N-2hydroxyethylpiperazine-N9-2-ethanesulfonic acid (HEPES; pH 7.9)–400 mM NaCl–0.1 mM EDTA–0.1 mM ethylene glycol-bis(b-aminoethyl ether)N,N,N9,N9-tetraacetic acid (EGTA)–2 mM dithiothreitol–1% Nonidet P-40–2 mg of pepstatin per ml–2 mg of leupeptin per ml–1 mg of aprotinin per ml. Western blot (immunoblot) analysis. A 20-ml volume of nuclear extract (approximately 2.5 mg/ml) was size fractionated on a sodium dodecyl sulfate (SDS)– polyacrylamide gel and transferred to nitrocellulose membranes. The nitrocellulose filters were blocked for 1 h at room temperature with 5% nonfat milk in PBST. After being rinsed with PBST, the membranes were incubated for 1 h at room temperature with rabbit anti-Tst-1 or anti-JCT polyclonal antiserum, each diluted 1:3,000 in PBST. Following three washes with PBST, the membranes were incubated for 20 min at room temperature with horseradish peroxidasecoupled protein A in PBST at a 1:3,000 dilution. After extensive washing, the antigen was detected with the enhanced chemiluminescence detection system (Amersham) as specified by the manufacturer.

RESULTS DNA replication of JC virus variants in glial and nonglial primate cells. Comparative sequence analysis reveals that most differences between regulatory regions from archetype and PML-type strains of JC virus are found in regions flanking the core origin of DNA replication. By contrast, the core origin itself, as defined in previous studies (33, 52, 53), is strongly conserved among the isolates. Therefore, it is not to be expected that these core regions would differ strongly from each other in their replicative ability. However, it has been shown that sequences which naturally flank the core origin have auxiliary functions in the replication process and can strongly influence the rate of DNA replication (33, 53). Therefore, it was of interest to compare the levels of DNA replication directed by each of the regulatory regions of various JC virus isolates. Regulatory regions of JC virus strains G2, N1, and MY (62) were chosen from the archetype group, while Mad-1 (15) and GS/B (32) represent two different PML-type strains. As previously shown, DNA replication from JC virus regulatory regions can be observed in primate cells only in the presence of the viral large T antigen (13). None of the cell lines used in this study expressed T antigen endogenously (Fig. 1 and data not shown), but they all could be induced to do so by

1513

transfection with pRSV-JCT, an expression plasmid in which the gene of large T antigen is under the control of the highly active Rous sarcoma virus long terminal repeat (52). Expression of JC virus T antigen could be visualized both in Western blots and in immunofluorescence studies with a rabbit polyclonal antiserum generated against recombinant JC virus T antigen produced in baculovirus (Fig. 1A and B). Furthermore, immunofluorescence studies reveal the predominantly nuclear localization of T antigen (Fig. 1B). For DNA replication studies, plasmids carrying virus regulatory regions were cotransfected with pRSV-JCT into U138 human glioblastoma or CV1 monkey kidney cells. DNA which had replicated in these cells during a period of 72 h was distinguished from DNA that had not by their differential sensitivity toward the restriction endonuclease DpnI. The DpnI-resistant newly replicated DNA was visualized and quantitated on a Southern blot. As shown in Fig. 1C, no DNA replication could be observed in the absence of T antigen or the origin of DNA replication. A plasmid devoid of an origin did not exhibit a significant amount of DNA replication even in the presence of T antigen (Fig. 1C, lane 2), while a plasmid carrying the regulatory region of Mad-1 replicated efficiently in the same cells, as long as T antigen was provided (compare lanes 1 and 3). Therefore, the replication assays also prove that the T antigen expressed from pRSV-JCT is not only full length and correctly localized within the cell but also functionally active. Only minor differences could be observed in the replication rates of the various archetype and PML-type isolates (Table 1). In general, replication rates from archetype regulatory regions were slightly higher in CV1 cells than were those conferred by PML types. This effect was not observed in U138 cells. Instead, the regulatory regions of archetypes G2 and MY exhibited a replication rate slightly lower than that observed for the PMLtype strains Mad-1 and GS/B. The regulatory region which consistently provided the highest replication rate was the one from the archetype N1. However, replication rates vary less than twofold between the various regulatory regions in any given cell. Thus, it is uncertain whether any of the observed differences are functionally important. Glia specificity of viral gene expression. In the past, various parts of the regulatory region of JC virus Mad-1 (and other PML-type strains) have been used to show the glia specificity of viral gene expression (13, 21, 25, 27, 56). Seldom, however, has the whole regulatory region between the open reading frames for both early and late genes been used in these studies. Therefore, it was of interest to test whether the whole regulatory region would still exhibit the same glia specificity that has been observed for shorter promoter fragments. Figure 2 shows the results of a representative study. To calculate the relative activity of JC virus promoters in glial versus nonglial cells, the expression of viral early and late promoters was normalized to expression levels from the cytomegalovirus (CMV) promoter in the same cell line. Similar results were obtained when a TATA box-containing basal promoter instead of the CMV promoter was used for normalization (data not shown). When the whole regulatory region was used, the early and late promoters of the prototypic strain Mad-1 were 16.5- to 11-fold more active in glial than in nonglial cells. Thus, while there is some glia specificity of both promoters, the effect is not as pronounced as for shorter fragments of the viral regulatory region (25). The regulatory region of GS/B, the second PMLtype isolate, exhibited a similar level of glia specificity, with the early and late promoters being approximately 18- and 15-fold more active, respectively, in glial than in nonglial cells. The same analysis was also performed on the regulatory

1514

SOCK ET AL.

J. VIROL.

FIG. 1. DNA replication of plasmids carrying whole regulatory regions of JC virus strains in the presence of ectopically expressed JC viral T antigen. (A) Western blot analysis. Extracts from CV1 cells transfected with pRSV-JCT (lane 4) were compared with extracts from untransfected CV1 cells (lane 3) by using a polyclonal rabbit antiserum raised against JC virus T antigen. Portions (10 ng) of recombinant SV40 and JC virus T antigen produced in baculovirus-infected insect cells were loaded as controls (lanes 1 and 2). The different mobilities of JC virus T antigens expressed in baculovirus and CV1 cells result from the presence of a histidine tag in the recombinant protein. The sizes of markers are given in kilodaltons. (B) Immunofluorescence of CV1 cells which were mock transfected (upper panel) or transfected with pRSV-JCT (lower panel) by using rabbit anti-JCT antiserum (diluted 1:500) as primary antibody and Cy3-conjugated goat anti-rabbit antibodies (diluted 1:500) as secondary antibodies. Magnification, 380. (C) DpnI sensitivity assay. Hirt extracts (53) from transfected U138 cells were cut with DpnI and BglII, size fractionated on a 1% agarose gel, blotted onto Hybond N1 membranes (Amersham), and hybridized to pGLbasic sequences (Promega): pGL-Mad1 (lane 1), pGLbasic plus pRSV-JCT (lane 2); pGL-Mad1 plus pRSV-JCT (lane 3); pGL-GS/B plus pRSV-JCT (lane 4); pGL-G2 plus pRSV-JCT (lane 5); pGL-N1 plus pRSV-JCT (lane 6); pGL-MY plus pRSV-JCT (lane 7).

regions of the three archetype strains G2, N1, and MY. Basal levels of expression were in the same range for promoters from archetype and PML-type strains, with activities varying less than 1 order of magnitude. Thus, the differences within the regulatory regions of archetype and PML-type strains do not dramatically affect the basal level of viral gene expression. Early and late promoters of all archetype regulatory regions proved to be more active in glial than in nonglial cells, as was already observed for the PML-type strains. However, small but significant differences in the degree of glia specificity between archetype and PML-type strains could be observed. Promoters from archetype strains were only approximately 5-fold more active in glial than in nonglial cells, compared with the 11- to 18-fold difference observed for the PML-type promoters. Influence of T antigen on viral gene expression. Gene expression in polyomaviruses has been shown to be regulated by one of the early genes, the large T antigen. For the related simian virus 40 (SV40), T antigen has been shown to inhibit transcription of early viral genes, thereby regulating its own

expression (1, 11, 18, 26, 40, 45, 50). At the same time, SV40 T antigen activates transcription of the late viral genes (7, 24). We have previously shown that JC virus T antigen also stimulates late-gene expression of prototype Mad-1 in U138 cells (49). However, we were unable to detect a strong T-antigendependent reduction of early-gene expression in these cells. Here we analyzed the influence of T antigen on early-gene expression for various archetype and PML-type strains in a number of glial and nonglial cell lines by using a very sensitive luciferase assay (Fig. 3). For all luciferase assays, cells were harvested 48 h after transfection. During that period, JC virus T antigen was able to direct only very small amounts of DNA replication in cells of primate origin whereas no DNA replication could be observed in rodent cell lines (data not shown). Thus, while the luciferase reporter plasmid can replicate in a T-antigen-dependent manner, this effect was negligible during the course of the luciferase assay. Early-gene expression of the prototype strain Mad-1 was largely unaffected by T antigen in various glial cells, including U138, U87, and 33B cells. The

VOL. 70, 1996


TABLE 1. DNA replication rates of regulatory regions from various strains of JC virus Cell type

Viral strain

Relative replication activitya

CV1

G2 N1 MY GS/B Mad-1

100.7 6 21.6 158.5 6 26.2 124.2 6 23.2 74.9 6 10.1 100

U138

G2 N1 MY GS/B Mad-1

80.7 6 18.7 103.1 6 19.3 67.9 6 14.1 95.0 6 12.5 100

a Replication activities of pGL-G2, pGL-N1, pGL-MY, pGL-GS/B, and pGLMad1 were determined in CV1 and U138 cells which had been cotransfected with pRSV-JCT. DpnI sensitivity assays were carried out as described previously (53), and DpnI-resistant, full-length plasmid DNA was quantified by PhosphorImager analysis of Southern blots. All values are expressed relative to the replication activity of pGL-Mad1, which was arbitrarily given a value of 100. Results represent the average activity 6 standard error of the mean as determined in four experiments.

activity of the early promoter of Mad-1 remained almost unchanged in U138 and U87 cells, and it was stimulated less than twofold in 33B cells. Significant inhibition of early-gene expression was, however, observed for Mad-1 in several nonglial cells and was most pronounced in mouse L tk2 cells. Importantly, all early promoters were inhibited by T antigen in a similar manner in L tk2 cells independently of whether they originated from an archetype or a PML-type strain. Thus, it must be concluded that T-antigen-dependent inhibition of viral earlygene expression is much more dependent on the cellular background than on the viral isolate. Similar results were obtained in the presence and absence of aphidicolin, an inhibitor of DNA polymerase a, again arguing against a major role of DNA replication in the luciferase assays (data not shown). Similar analyses were also performed to quantitate the effect of T antigen on viral late-gene expression (Fig. 4). Differences among various cell lines were more pronounced than differences among the various viral isolates. T-antigen stimulated viral late-gene expression in all cells analyzed. However, while this stimulation was only 2- to 3-fold on average in rat 33B and

1515

mouse L tk2 cells, several primate cells exhibited much higher stimulation rates, sometimes reaching a 10-fold or higher stimulation. The responsiveness of viral late promoters toward T antigen was generally as high in archetype strains as it was in PML-type strains. Again, similar results were obtained in the presence and absence of aphidicolin. No significant amount of DNA replication could be observed during the 48-h period of the assay (data not shown). Thus, stimulation of viral late-gene expression was dependent on the cell type, with cells of primate origin exhibiting a higher response than cells of rodent origin. Influence of Tst-1/Oct6 on viral gene expression. We have previously shown that both early- and late-gene expression of JC virus Mad-1 can be stimulated by the POU domain transcription factor Tst-1/Oct6, which is present in several glial cells that are naturally targeted by JC virus (49, 60). The response of viral gene expression to a glial transcription factor could therefore be a decisive difference between archetype and PML-type strains. To investigate this question in greater detail, we examined the influence of Tst-1/Oct6 on viral early- and late-gene promoters in a series of different cellular contexts. As a first step in this analysis, we tried to clarify whether any of the cells used in our study expressed Tst-1/Oct6 endogenously. Like T antigen, Tst-1/Oct6 is a nuclear protein (28). Figure 5 shows a Western blot of nuclear extracts from several glial and nonglial cell lines. As a positive control, we chose an extract from cells transiently transfected with an expression plasmid for Tst-1/Oct-6. With this extract, a strong immunoreactive band of 47 kDa was visualized. No Tst-1/Oct6 could be detected in U138 cells, in agreement with previous Northern (RNA) blot analyses (60). Similarly, neither U87, CV1, L tk2, nor 293 cells expressed significant amounts of this transcription factor. Endogenous Tst-1/Oct6 could be detected only in the rat oligodendroglioma cell line 33B. No specific immunoreactivity could be detected in cytosolic extracts of the same cell lines (data not shown). When tested for its influence on viral early-gene expression, Tst-1/Oct6 could stimulate the early promoter of the prototype strain Mad-1 in all cell lines of glial origin (Fig. 6). In good agreement with previously published results (49, 60), stimulation rates were approximately 6.5-fold on average in U138 cells. Compared with U138 cells, U87 cells exhibited a slightly lower, 5.8-fold stimulation of Mad-1 early-gene expression, whereas the stimulation was even more robust in 33B cells (12.4-fold). Thus, the activity of Tst-1/Oct6 was highest in cells

FIG. 2. Glia specificity of gene expression for various JC virus strains. Early-gene expression (A) and late-gene expression (B) were analyzed for their glia specificity by determining the activity of each archetype and PML-type promoter in U138 human glioblastoma and 293 human embyonic kidney cells relative to the activity of the CMV promoter/enhancer region. Relative activities were then expressed as the ratio of glial versus nonglial activity. Similar results were obtained in three independent experiments as well as in comparisons with other glial and nonglial cell lines.

1516

SOCK ET AL.

J. VIROL.

the case of Mad-1, ranging from a 6.5-fold stimulation in U87 cells to a 7.6-fold stimulation in U138 cells to an 11.1-fold stimulation in 33B cells. Similarly, late-gene promoters of the archetypes N1 and MY were strongly stimulated by Tst-1/Oct6 in primate and rodent glial cells. The late promoter of strain N1, for instance, was stimulated on average 5.7-fold in U87 cells, 11.9-fold in U138 cells, and 24.5-fold in 33B cells. Again, strain GS/B represented the exception, with its late-gene promoter being as unresponsive to Tst-1/Oct6 as its early promoter. No stimulation of late-gene promoter activity by Tst-1/ Oct6 could be observed in nonglial cells. The activity of all late-gene promoters was inhibited by Tst-1/Oct6 in L tk2 cells, lending further support to the glia specificity of Tst-1/Oct6 function. DISCUSSION It has been noted previously that JC virus strains show a significant natural variance (5, 14, 23, 39, 58, 61, 63). Differ-

FIG. 3. Influence of JC virus T antigen on early-gene expression of JC virus strains. Transient transfections were carried out in U138, U87, 33B, CV1, and L 2 tk cells. Luciferase reporter plasmids carrying various virus regulatory regions in early orientation were transfected into these cells with or without the expression plasmid for JC virus T antigen, pRSV-JCT. The following reporter plasmids were used: pGL-G2early (G2), pGL-N1early (N1), pGL-MYearly (MY), pGL-GS/ Bearly (GS/B), and pGL-Mad1early (Mad-1). Luciferase activities in extracts from transfected cells were determined in at least three independent experiments, each performed in duplicate. Data are presented as fold inhibitions, which were calculated for each reporter plasmid by comparing values from cells transfected with expression plasmid pRSV-JCT with values from cells transfected with empty expression plasmid pRSV.

which also endogenously express the protein. In contrast to glial cells, neither monkey kidney (CV1), human kidney (293), nor mouse fibroblast (L tk2) cells exhibited a significant Tst1/Oct6-dependent stimulation of the early promoter of Mad-1. Rather, L tk2 and 293 cells exhibited a significant two- to fourfold repression of early-gene expression in the presence of ectopically expressed Tst-1/Oct6. This cell-type-specific function is most probably due to the glia specificity of Tst-1/Oct6 (see Discussion). A Tst-1/Oct6-dependent stimulation of earlygene expression was also observed for all archetype strains in glial cells. In 33B cells, Tst-1/Oct6 activated early-gene expression of all archetypes even more strongly than for Mad-1. Interestingly, early-gene expression of the PML-type strain GS/B was largely unresponsive to Tst-1/Oct6 in both glial and nonglial cells (see Discussion). Analogous transfection analyses were also carried out for late-gene expression (Fig. 7). In glial cells, Tst-1/Oct6 also induced a robust induction of late-gene promoter activity in

FIG. 4. Influence of JC virus T antigen on late-gene expression of JC virus strains. Transient transfections were carried out in U138, U87, 33B, CV1, and L tk2 cells. Luciferase reporter plasmids carrying various virus regulatory regions in late orientation were transfected into these cells with or without pRSV-JCT. The following reporter plasmids were used: pGL-N1late (N1), pGL-MYlate (MY), pGL-GS/Blate (GS/B), and pGL-Mad1late (Mad-1). Luciferase activities in extracts from transfected cells were determined in at least three independent experiments, each performed in duplicate. Data are presented as fold activations, which were calculated for each reporter plasmid by comparing values from cells transfected with expression plasmid pRSV-JCT with values from cells transfected with empty expression plasmid pRSV.

VOL. 70, 1996


FIG. 5. Expression of Tst-1/Oct6 in various glial and nonglial cell lines. Nuclear extracts of U138 human glioblastoma (lane 1), U87 human glioblastoma (lane 2), 33B rat oligodendroglioma (lane 3), CV1 monkey kidney cells (lane 4), mouse L tk2 cells (lane 5), and human kidney 293 cells (lane 6) were separated on SDS–10% polyacrylamide gels. Tst-1/Oct6 was detected by using a polyclonal antiserum against Tst-1/Oct6 as primary antibody and horseradish peroxidasecoupled protein A (both diluted 1:3,000) as secondary antibody. An extract from CV1 cells transiently transfected with pCMV/Tst-1 served as a control (lane 7). Sizes of markers are indicated on the left of the gel in kilodaltons.

ences are mainly clustered in the hypervariable viral regulatory region. This region is centered between the two viral transcription units and contains the origin of viral DNA replication as well as all transcriptional regulatory sequences. Thus, it effectively controls viral gene expression and genome multiplication (16, 35). JC virus isolates are usually classified as either PML type or archetype. It has been postulated that PML-type strains arise from archetype strains by a series of genomic rearrangements, although this model has yet to be experimentally proven. As a result of these rearrangements, the nonpathogenic archetype form is believed to be transformed into the pathogenic PML type, which now possesses altered tissue specificity or has gained oncogenic potential (16, 23, 32, 39, 63). The high regulatory potential of the viral regulatory region argues in favor of such a model. Alternatively, the archetype is already competent to infect central nervous system glia without the need for preceding rearrangements in its regulatory region. Rearrangements that occur would then represent an optimized adaptation to the environment of its host oligodendrocyte. Therefore, the key question is whether a switch from an archetype to a PML-type strain is an important step in the pathogenesis of PML or whether it represents only a late consequence of the disease. This study was undertaken to shed some light on this question. Regulatory regions from several archetype and PML-type strains of JC virus were compared with respect to various functions. Only minor differences were observed in the ability of the various regulatory regions to direct DNA replication in the presence of JC virus T antigen in primate cells. Archetype regulatory regions were slightly more active than PML-type regions in the monkey kidney cell line CV1, whereas they showed a slightly lower activity in the glioblastoma cell line U138. However, effects were fairly subtle, with replication rates diverging no more than a factor of 2 between various regulatory regions. This result might have been expected because of the strong sequence conservation of the core origin of DNA replication (33, 34, 52, 53). Nevertheless, flanking sequences, though not essential for DNA replication, have a strong modulatory function on core origin activity, as previously shown for JC virus strain Mad-1 (33, 52, 53) or, more extensively, for the related SV40 (10, 17, 22, 30, 31). Thus, it is

1517

noteworthy that the significant differences in the origin-flanking regions of archetype and PML-type strains do not translate into significantly altered replication rates. Surprising results were obtained when the tissue specificity of archetype and PML-type gene expression was analyzed. Although less pronounced than in PML-type strains, both early- and late-gene expression from archetype regulatory regions exhibited an increased rate in glial cells compared with nonglial cells. This glia specificity of archetype gene expression argues against a scenario in which a conversion from archetype to PML type would be needed for the initial infection of oligodendroglia during the onset of PML. Rearrangements would then have to occur in later stages of the infection, leading to the selection of a PML-type strain whose gene expression is even better adapted to the glial environment than the original archetype. Our findings are supported by recent studies (21, 27) which show that the minimal early-gene promoter of JC virus already displays some degree of glia specificity. This minimal promoter is strongly preserved in all archetype isolates

FIG. 6. Influence of Tst-1/Oct6 on early-gene expression of JC virus strains. Transient transfections were carried out in U138, U87, 33B, CV1, L tk2, and 293 cells. Luciferase reporter plasmids carrying various virus regulatory regions in early orientation were transfected into these cells with or without the expression plasmid for Tst-1/Oct6, pCMV/Tst-1. The following reporter plasmids were used: pGL-G2early (G2), pGL-N1early (N1), pGL-MYearly (MY), pGL-GS/Bearly (GS/ B), and pGL-Mad1early (Mad-1). Luciferase activities in extracts from transfected cells were determined in at least three independent experiments, each performed in duplicate. Data are presented as fold stimulations, which were calculated for each reporter plasmid by comparing values from cells transfected with expression plasmid pCMV/Tst-1 with values from cells transfected with empty CMV expression plasmid.

1518

SOCK ET AL.

FIG. 7. Influence of Tst-1/Oct6 on late-gene expression of JC virus strains. Transient transfections were carried out in U138, U87, 33B, CV1, L tk2, and 293 cells. Luciferase reporter plasmids carrying various virus regulatory regions in late orientation were transfected into these cells with or without pCMV/Tst-1. The following reporter plasmids were used: pGL-N1late (N1), pGL-MYlate (MY), pGL-GS/Blate (GS/B), and pGL-Mad1late (Mad-1). Luciferase activities in extracts from transfected cells were determined in at least three independent experiments, each performed in duplicate. Data are presented as fold activations, which were calculated for each reporter plasmid by comparing values from cells transfected with expression plasmid pCMV/Tst-1 with values from cells transfected with empty CMV expression plasmid.

(63). Its unaltered presence could therefore be one of the reasons why whole regulatory regions from archetype strains drive viral gene expression in a glia-specific manner. Gene expression of JC virus Mad-1 has been shown in the past to be modulated by the absence or presence of a whole series of transcriptional regulators in a given cell (2–4, 20, 21, 29, 46, 48, 49, 57, 60). One of these proteins is the POU domain transcription factor Tst-1/Oct6, which is also known as SCIP (19, 41, 44, 55). Among other cells, Tst-1/Oct6 is expressed in oligodendroglial cells of the central nervous system (9, 19) and has been implicated in regulating JC virus gene expression. It both binds to the regulatory region of the prototype strain Mad-1 and activates viral early- and late-gene promoters which are contained within this region (27, 49, 60). Here we analyzed the transcriptional response of various JC virus isolates to Tst-1/Oct6 in cotransfection experiments. Except for the oligodendroglioma cell line 33B, none of the other cell lines used in this study endogenously expressed significant

J. VIROL.

amounts of Tst-1/Oct6. In glial cells, both early- and late-gene promoters of all archetype strains were strongly stimulated by Tst-1/Oct6 independently of whether these cells express endogenous Tst-1/Oct-6. In sharp contrast to all glial cells, none of the other cell lines used in this study exhibited a significant stimulation by Tst-1/Oct6. While Tst-1/Oct6 had virtually no effect in CV-1 cells, it even inhibited gene expression in L tk2 and 293 cells. These findings are in good agreement with previous results, which show that Tst-1/Oct-6 functions much better in glial than in nonglial cells (27, 42, 43). Thus, while cells of different origin respond differently to Tst-1/Oct6, archetype strains of JC virus behave very similarly to the prototypic strain Mad-1. This similarity was not necessarily expected, despite the conservation of a functional Tst-1-binding site between Mad-1 and the archetypes. However, while Mad-1 carries two copies of this functionally relevant site as part of a larger 98-bp tandem repeat, all archetypes contain only one copy. In addition, Mad-1 and archetypes are different with respect to the environment in which this binding site is embedded. GS/B, the second PML-type viral isolate used in this study, had the weakest response to Tst-1/Oct6 and was at best slightly stimulated. Thus, differences between the two PML-type strains were more pronounced than differences between archetype and PML-type strains. Curiously, the Tst-1-binding site which was identified in Mad-1 is also present in the genome of GS/B. Although the flanking sequences for this site differ between Mad-1 and GS/B, they are identical between GS/B and the archetypes, which are responsive to Tst-1/Oct6. At present, we cannot fully explain this difference between GS/B and all other viral isolates. Most probably, however, Tst-1 function in the context of the viral regulatory region involves multiple interactions with other transcription factors which also interact with this region. Given the intactness of the Tst-1/Oct6-binding site in the GS/B genome, disruption of one of these interactions seems to be a plausible explanation for the different behavior of GS/B. We were also interested in whether archetype and PML-type viral strains would differ from each other by their response to the multifunctional viral early-gene product T antigen (for a review of the related SV40 T antigen, see reference 12). Besides its function in viral DNA replication, T antigen functions as a transcriptional regulator (21, 29, 46, 49, 56). As already observed for Tst-1/Oct6, differences in the influence of T antigen on transcription were much more pronounced between different cell types than between archetype and PML-type strains. If a significant effect of JC virus T antigen on earlygene expression could be detected, it was an inhibitory one. Inhibition of early-gene expression could not be observed in any glial cell line but was readily observed for Mad-1 in CV1 cells and for all strains in L tk2 cells. Transcriptional effects of T antigen were measured 2 days after transfection. During this period, no significant amount of DNA replication could be detected. Thus, these transcriptional effects of T antigen were clearly separate from its functions in DNA replication. This interpretation is also supported by the fact that similar results were obtained in the presence and absence of the DNA synthesis inhibitor aphidicolin. In any case, JC virus T antigen does not seem to be as strong an inhibitor of viral early-gene expression as is SV40 T antigen (45, 50). Furthermore, the strength of the inhibitory function seems to be dependent on the cellular background and seems to be alleviated in glial cells, probably because of the productive interaction of T antigen with glial proteins. It has been shown, for instance, that the combination of JC virus T antigen and Tst-1/Oct6 can synergistically stimulate early-gene expression in glial cells (49).

VOL. 70, 1996


In contrast to early-gene transcription, viral late-gene transcription was always stimulated by the presence of JC virus T antigen. Stimulation rates were consistently highest in cell lines of primate origin. The two cell lines of rodent origin exhibited only a marginal stimulation of T-antigen-dependent late-gene expression. This might point to a species-specific component of the late function of JC virus T antigen. Such species specificity was not detected for the corresponding early function. Again, no major difference could be detected in the response of archetype and PML-type strains with regard to T-antigen-dependent stimulation of late-gene transcription. The present study aims at comparing several prominent features of the regulatory regions from PML-type and archetype strains in cultures of established cell lines. It does not address the issue of whether archetype sequences can direct lytic infection and multiplication of JC virion particles. Although our data have yet to be confirmed in primary cell cultures, they clearly indicate that regulatory regions from archetype and PML-type strains of JC virus are remarkably similar with regard to their replication and transcription functions. The small differences in replication or transcription rates which can be detected between them are compatible with a model in which the PML-type strains of JC virus are better adapted to their glial environment than are their archetype counterparts. Nevertheless, it seems clear that all crucial aspects of gene regulation and DNA replication which have been analyzed in this set of experiments are conserved between the two forms of viral strains. This also includes the glia specificity of viral gene expression, which is clearly already detectable in the archetype strains. Thus, our results are most compatible with a model in which archetype strains do not have to undergo a conversion to PML-type strains to be able to infect glial cells of the central nervous system. Conversion would, rather, be a consequence of infection. ACKNOWLEDGMENTS We thank J. Gerber and F. Grummt for the anti-JCT antiserum and Y. Yogo and K. Do ¨rries for the JC virus strains G2, N1, MY, and GS/B, respectively. This work was supported by a grant from the Wilhelm SanderStiftung (93.066.1) to M.W.

11. 12. 13. 14.

15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28.

29. REFERENCES 1. Alwine, J. C., S. I. Reed, and G. R. Stark. 1977. Characterization of the autoregulation of simian virus 40 gene. J. Virol. 24:22–27. 2. Amemiya, K., R. Traub, L. Durham, and E. O. Major. 1989. Interaction of a nuclear factor-1-like protein with the regulatory region of the human polyomavirus JC virus. J. Biol. Chem. 264:7025–7032. 3. Amemiya, K., R. Traub, L. Durham, and E. O. Major. 1992. Adjacent nuclear factor-1 and activator protein binding sites in the enhancer of the neurotropic JC virus. J. Biol. Chem. 267:14204–14211. 4. Atwood, W. J., L. Wang, L. C. Durham, K. Amemiya, R. G. Traub, and E. O. Major. 1995. Evaluation of the role of cytokine activation in the multiplication of JC virus (JCV) in human fetal glial cells. J. NeuroVirol. 1:40–49. 5. Ault, G. S. 1993. Human polyomavirus JC promoter enhancer rearrangement patterns from progressive multifocal leukoencephalopathy brain are unique derivatives of a single archetypal structure. J. Gen. Virol. 74:1499– 1507. 6. Berger, J. R., B. Kaszovitz, J. D. Post, and G. Dickinson. 1987. Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection: a review of the literature with a report of sixteen cases. Ann. Intern. Med. 107:78–87. 7. Brady, J. N., J. B. Bolen, M. Radonovich, N. Salzman, and G. Khoury. 1984. Stimulation of simian virus 40 late expression by simian virus 40 tumor antigen. Proc. Natl. Acad. Sci. USA 81:2040–2044. 8. Chen, C., and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745–2752. 9. Collarini, E. J., R. Kuhn, C. J. Marshall, E. S. Monuki, G. Lemke, and W. D. Richardson. 1992. Down-regulation of the POU transcription factor SCIP is an early event in oligodendrocyte differentiation in vitro. Development 116: 193–200. 10. DeLucia, A. L., S. Deb, K. Partin, and P. Tegtmeyer. 1986. Functional

30.

31.

32. 33. 34. 35.

36.

37.

38.

39.

1519

interactions of the simian virus core origin of replication with flanking regulatory sequences. J. Virol. 57:138–144. DiMaio, D., and D. Nathans. 1982. Regulatory mutants of simian virus 40. J. Mol. Biol. 156:531–548. Fanning, E. 1992. Simian virus 40 large T antigen: the puzzle, the pieces, and the emerging picture. J. Virol. 66:1289–1293. Feigenbaum, L., K. Khalili, E. Major, and G. Khoury. 1987. Regulation of the host range of human papovavirus JCV. Proc. Natl. Acad. Sci. USA 84: 3695–3698. Flaegstad, T., A. Sundsfjord, R. R. Arthur, M. Pedersen, T. Traavik, and S. Subramani. 1991. Amplification and sequencing of the control regions of BK and JC virus from human urine by polymerase chain reaction. Virology 180: 553–560. Frisque, R. J., G. L. Bream, and M. T. Cannella. 1984. Human polyomavirus JC virus genome. J. Virol. 51:458–469. Frisque, R. J., and F. A. White III. 1992. The molecular biology of JC virus, causative agent of progressive multifocal leukoencephalopathy, p. 25–158. In R. Ross (ed.), Molecular neurovirology. Humana Press, Clifton, N.J. Guo, Z. S., C. Gutierrez, U. Heine, J. M. Sogo, and M. L. De Pamphilis. 1989. Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo. Mol. Cell. Biol. 9:3593–3602. Hansen, U., D. G. Tenen, D. M. Livingston, and P. A. Sharp. 1981. T antigen repression of SV40 early transcription from two promoters. Cell 27:603–612. He, X., R. Gerrero, D. M. Simmons, R. E. Park, C. R. Lin, L. W. Swanson, and M. G. Rosenfeld. 1991. Tst-1, a member of the POU domain gene family, binds the promoter of the gene encoding the cell surface adhesion molecule P0. Mol. Cell. Biol. 11:1739–1744. Henson, J. W. 1994. Regulation of the glial-specific JC virus early promoter by the transcription factor Sp1. J. Biol. Chem. 269:1046–1050. Henson, J. W., B. L. Schnitker, T.-S. Lee, and J. McAllister. 1995. Cellspecific activation of the glial-specific JC virus early promoter by large T-antigen. J. Biol. Chem. 270:13240–13245. Hertz, G. Z., and J. E. Mertz. 1986. Bidirectional promoter elements of simian virus 40 are required for efficient replication of the viral DNA. Mol. Cell. Biol. 6:3513–3522. Iida, T., T. Kitamura, J. Guo, F. Taguchi, Y. Aso, K. Nagashima, and Y. Yogo. 1993. Origin of JC polyomavirus variants associated with progressive multifocal leukoencephalopathy. Proc. Natl. Acad. Sci. USA 90:5062–5065. Keller, J. M., and J. C. Alwine. 1984. Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication. Cell 36: 381–389. Kenney, S., V. Natarajan, D. Strike, G. Khoury, and N. P. Salzman. 1984. JC virus enhancer-promoter active in human brain cells. Science 226:1337–1339. Khoury, G., and E. May. 1977. Regulation of early and late simian virus 40 transcription: overproduction of early viral RNA in the absence of functional T antigen. J. Virol. 23:167–176. Krebs, C. J., M. T. McAvoy, and G. Kumar. 1995. The JC virus minimal core promoter is glial cell specific in vivo. J. Virol. 69:2434–2442. Kuhn, R., E. S. Monuki, and G. Lemke. 1991. The gene encoding the transcription factor SCIP has features of an expressed retroposon. Mol. Cell. Biol. 11:4642–4650. Lashgari, M., H. Tada, S. Amini, and K. Khalili. 1989. Regulation of JCVL promoter function. Transactivation of JCVL promoter by JCV and SV40 early proteins. Virology 170:292–295. Lee-Chen, G. J., and M. Woodworth-Gutai. 1986. Simian virus 40 DNA replication: functional organization of regulatory elements. Mol. Cell. Biol. 6:3086–3093. Li, J. J., P. W. C. Peden, R. A. F. Dixon, and T. Kelly. 1986. Functional organization of the simian virus 40 origin of DNA replication. Mol. Cell. Biol. 6:1117–1128. Loeber, G., and K. Do¨rries. 1988. DNA rearrangements in organ-specific variants of polyomavirus JC strain GS. J. Virol. 62:1730–1735. Lynch, K. J., and R. J. Frisque. 1990. Identification of critical elements within the JC virus DNA replication origin. J. Virol. 64:5812–5822. Lynch, K. J., and R. J. Frisque. 1991. Factors contributing to the restricted DNA replicating activity or JC virus. Virology 180:306–317. Major, E. O., K. Amemiya, C. S. Tornatore, S. A. Houff, and J. R. Berger. 1992. Pathogenesis and molecular biology of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain. Clin. Microbiol. Rev. 5:49–73. Major, E. O., A. E. Miller, P. Mourrain, R. G. Traub, E. DeWidt, and J. Sever. 1985. Establishment of a line of human fetal glial cells that supports JC virus multiplication. Proc. Natl. Acad. Sci. USA 82:1257–1261. Mandl, C., D. L. Walker, and R. J. Frisque. 1987. Derivation and characterization of POJ cells, transformed human fetal glial cells that retain the permissivity for JC virus. J. Virol. 61:755–763. Markowitz, R.-B., B. A. Eaton, M. F. Kubik, D. Latorra, J. A. McGregor, and W. S. Dynan. 1991. BK virus and JC virus shed during pregnancy have predominantly archetypal regulatory regions. J. Virol. 65:4515–4519. Martin, J. D., D. M. King, J. M. Slauch, and R. J. Frisque. 1985. Differences in regulatory sequences of naturally occurring JC virus variants. J. Virol. 53: 306–311.

1520

SOCK ET AL.

40. McKnight, S., and R. Tjian. 1986. Transcriptional selectivity of viral genes in mammalian cells. Cell 46:795–805. 41. Meijer, D., A. Graus, R. Kraay, A. Langeveld, M. P. Mulder, and G. Grosveld. 1990. The octamer binding factor Oct6: cDNA cloning and expression in early embryonic cells. Nucleic Acids Res. 18:7357. 42. Monuki, E. S., R. Kuhn, and G. Lemke. 1993. Cell-specific action and mutable structure of a transcription factor effector domain. Proc. Natl. Acad. Sci. USA 90:9978–9982. 43. Monuki, E. S., R. Kuhn, and G. Lemke. 1993. Repression of the myelin P0 gene by the POU transcription factor SCIP. Mech. Dev. 42:15–32. 44. Monuki, E. S., G. Weinmaster, R. Kuhn, and G. Lemke. 1989. SCIP: a glial POU domain gene regulated by cyclic AMP. Neuron 3:783–793. 45. Myers, R. M., C. R. Conald, A. K. Robbins, and T. Robert. 1981. SV40 gene expression is modulated by the cooperative binding of T antigen to DNA. Cell 25:373–384. 46. Nakshatri, H., A. Pater, and M. M. Pater. 1990. Activity and enhancer binding factors for JC virus regulatory elements in differentiating embryonal carcinoma cells. Virology 177:784–789. 47. Padgett, B. L., and D. L. Walker. 1973. Prevalence of antibodies in human sera against JC virus, an isolate from a case of progressive multifocal leukoencephalopathy. J. Infect. Dis. 127:467–470. 48. Ranganathan, P. N., and K. Khalili. 1993. The transcriptional enhancer element, kB, regulates promoter activity of the human neurotropic virus, JCV, in cells derived from the CNS. Nucleic Acids Res. 21:1959–1964. 49. Renner, K., H. Leger, and M. Wegner. 1994. The POU domain protein Tst-1 and papovaviral large tumor antigen function synergistically to stimulate glia-specific gene expression of JC virus. Proc. Natl. Acad. Sci. USA 91: 6433–6437. 50. Rio, D., A. Robbins, R. Myers, and R. Tjian. 1980. Regulation of simian virus 40 transcription in vitro by a purified tumor antigen. Proc. Natl. Acad. Sci. USA 77:5706–5710. 51. Schreiber, E., P. Matthias, M. M. Mu ¨ller, and W. Schaffner. 1989. Rapid detection of octamer binding proteins with ‘mini-extracts’ prepared from a small number of cells. Nucleic Acids Res. 17:6419. 52. Sock, E., M. Wegner, E. A. Fortunato, and F. Grummt. 1993. Large Tantigen and sequences within the regulatory region of JC virus both contribute to the features of JC virus DNA replication. Virology 197:537–548.

J. VIROL. 53. Sock, E., M. Wegner, and F. Grummt. 1991. DNA replication of human polyomavirus JC is stimulated by NF-1 in vivo. Virology 182:298–308. 54. Stoner, G. L., C. F. Ryschkewitsch, D. L. Walker, and H. D. Webster. 1986. JC papovavirus large tumor (T)-antigen expression in brain tissue of acquired immune deficiency syndrome (AIDS) and non-AIDS patients with progressive multifocal leukoencephalopathy. Proc. Natl. Acad. Sci. USA 83: 2271–2275. 55. Suzuki, N., H. Rohdewohld, T. Neumann, P. Gruss, and H. R. Scho¨ler. 1990. Oct-6: a POU transcription factor expressed in embryonal stem cells and in the developing brain. EMBO J. 9:3723–3731. 56. Tada, H., M. Lashgari, J. Rappaport, and K. Khalili. 1989. Cell type-specific expression of JC virus early promoter is determined by positive and negative regulation. J. Virol. 63:463–466. 57. Tamura, T., T. Inoue, K. Nagata, and K. Mikoshiba. 1988. Enhancer of human polyoma JC virus contains nuclear factor I-binding sequences: analysis using mouse brain nuclear extracts. Biochem. Biophys. Res. Commun. 157:419–425. 58. Tominaga, T., Y. Yogo, T. Kitamura, and Y. Aso. 1992. Persistence of archetypal JC virus DNA in normal renal tissue derived from tumor-bearing patients. Virology 186:736–741. 59. Weber, T., R. W. Turner, S. Frye, B. Ruf, J. Haas, E. Schielke, H. D. Pohle, W. Lueke, W. Lueer, K. Felgenhauer, and G. Hunsmann. 1994. Specific diagnosis of progressive multifocal leukoencephalopathy by polymerase chain reaction. J. Infect. Dis. 169:1138–1141. 60. Wegner, M., D. W. Drolet, and M. G. Rosenfeld. 1993. Regulation of JC virus by the POU-domain transcription factor Tst-1, implications for progressive multifocal leukoencephalopathy. Proc. Natl. Acad. Sci. USA 90:4743–4747. 61. White, F. A., M. Ishaq, G. L. Stoner, and R. J. Frisque. 1992. JC virus DNA is present in many human brain samples from patients without progressive multifocal leukoencephalopathy. J. Virol. 66:5726–5734. 62. Yogo, Y., T. Iida, F. Taguchi, T. Kitamura, and Y. Aso. 1991. Typing of human polyomavirus JC virus on the basis of restriction fragment length polymorphisms. J. Clin. Microbiol. 29:2130–2138. 63. Yogo, Y., T. Kitamura, C. Sugimoto, T. Ueki, Y. Aso, K. Hara, and F. Taguchi. 1990. Isolation of a possible archetypal JC virus DNA sequence from nonimmunocompromised individuals. J. Virol. 64:3139–3143.