Interactions between c-Jun, Nuclear Factor 1, and JC Virus Promoter Sequences: Implications for Viral Tropism^▿

Human primary tonsillar stromal cells (stromal cells).

Stromal cells were utilized due to their ability to support the complete JCV infectious cycle (24, 25) and high expression levels of NF-1 message (26). Cells were isolated according to protocols described previously (20).

Stromal protein extract.

Stromal cells were washed three times with ice-cold phosphate-buffered saline (PBS), and whole-cell extracts were prepared by a modification of the procedure of Andrews and Faller (3). Cells were scraped from tissue culture flasks, pelleted, and then disrupted by three freeze-thaw cycles on dry ice. Cell pellets were resuspended in two volumes of ice-cold buffer C (20 mM Tris-HCl [pH 7.9], 1.5 mM MgCl₂, 420 mM NaCl, 0.2 M EDTA, 25% glycerol) including protease inhibitors (0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 μg/μl antipain, 5 μg/μl leupeptin, 5 μg/μl chymostatin, 5 μg/μl aprotinin, 5 μg/μl pepstatin A). Lysates were then centrifuged at 9,000 × g for 5 min at 4°C. Supernatant protein fractions were divided into aliquots and stored at −80°C. Protein concentrations were determined by Bradford assay (5).

Radiolabeled oligonucleotide probes.

Oligonucleotides derived from JCV Mad-1 promoter sequence (nucleotides [nt] 38 to 53) (9) containing either an intact NF-1 binding site (5′-ATGGCTGCCAGCCAAG-3′) or a mutated NF-1 site (mutated sites underlined; 5′-ATTACTGCCAGCTGAG-3′) were synthesized (Invitrogen). Complementary strands were produced and annealed to each corresponding sequence above to form double-stranded oligonucleotides. The above double-stranded oligonucleotides (20 pM) were end labeled with [γ-³²P]ATP for 2 h at 37°C, centrifuged through a Microspin G-25 column (Pharmacia/GE Healthcare-Amersham Biosciences) at 735 × g for 2 min, and brought to a working concentration of 0.2 pM with buffer D (10 mM HEPES, 50 mM KCl, 10% glycerol).

Electrophoretic mobility shift assay (EMSA).

Unless otherwise noted, radiolabeled oligonucleotide probes (200,000 cpm) were incubated with 10 μg of stromal protein extract or 0.6 μg of recombinant human c-Jun [rhAP-1(c-Jun); amount recommended by the manufacturer; Promega] in the presence or absence of a 250-fold excess of either unlabeled intact oligonucleotide or unlabeled mutant oligonucleotide. As a binding control, one gel shift unit of the p50 subunit of recombinant human NF-κB was used (amount suggested by the manufacturer; Promega). Unless otherwise noted, incubations were carried out on ice for 30 min. Secondary incubations were conducted, where needed, by adding either cellular protein extract or rhAP-1(c-Jun) to the primary reaction mixture and then placing on ice for an additional 30 min. All incubations were resolved by electrophoresis on 6% polyacrylamide-Tris-glycine gels at 4°C. Gels were dried and exposed to BioMAX MR film (Kodak) for autoradiographical detection of protein/oligonucleotide binding as evidenced by any increase in the molecular weight of the probe (gel shift).

Human progenitor-derived astrocytes (PDA).

Human CNS progenitor cells (progenitors) were isolated from the telencephalon of a human fetal brain at 8 weeks of gestation, obtained in accordance to NIH guidelines and differentiated into an astrocytic lineage (PDA) according to a previously described protocol (26).

Immunoprecipitation (IP) and Western blot.

In these assays we utilized whole-cell extracts. Stromal and PDA cell cultures were lysed in ice-cold lysis buffer E (20 mM HEPES [pH 7.5], 150 mM NaCl, 3% glycerol, 5 mM MgCl₂, 1 mM CaCl₂, 1 mM EDTA, 1.0% NP-40, and one Complete Mini protease/phosphatase inhibitor tablet [Roche] per 20 ml). Lysates were sonicated on ice three times, 5 s each. After lysates were centrifuged to remove cellular debris, supernatants were precleared by incubation at 4°C for 1 h with 100 μl of protein G slurry (50% protein G agarose beads [Santa Cruz Biotechnology] suspended in 50% lysis buffer E) and 1 μg of a nonspecific rabbit polyclonal antibody (anti-14-3-3; Santa Cruz Biotechnology). Nonspecific complexes were pelleted by centrifugation at 10,000 × g at 4°C for 10 min, and the precleared supernatants were rotated at 4°C with either 1 μg of preimmune rabbit sera (Santa Cruz Biotechnology), anti-NF-1X (Geneka/Active Motif), or anti-c-Jun (Santa Cruz Biotechnology). After 1 h of incubation with these antibodies, a fresh 100 μl of protein G slurry was added for another 1 h of rotation at 4°C. The protein G beads were pelleted by centrifugation at 4°C and washed three times in ice-cold lysis buffer E, one time in ice-cold PBS. Beads were again pelleted by centrifugation at 4°C but then resuspended in 50 μl Laemmli protein loading buffer (PLB) and boiled for 5 min. Supernatants (the immunoprecipitates) from the pelleted beads were resolved on 12% precast sodium dodecyl sulfate (SDS)-polyacrylamide ReadyGels (Bio-Rad) and transblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore). The transblotted membranes were probed with primary antibodies (anti-c-Jun [Santa Cruz Biotechnology] or anti-NF-1X [Geneka/Active Motif]) at 4°C overnight in blocking buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk). Bound primary antibodies were detected with an antirabbit, horseradish peroxidase-conjugated secondary antibody and the Chemiluminescence Luminol reagent kit (Santa Cruz Biotechnology), according to the manufacturer's protocol.

Plasmid constructs.

A GST fusion protein plasmid, pGEX-Jun (21), which encodes GST-c-JUN, was generously provided by James A. Goodrich (University of Colorado). Additionally, pGEX2T, which encodes GST alone, was purchased (Pharmacia/GE Healthcare-Amersham Biosciences) as a suitable control plasmid (vector alone) for experiments conducted with the pGEX-Jun plasmid described above. The pcDNA3NF-1X plasmid was subcloned by forced cloning of the hemagglutinin (HA)-tagged NF-1X2 gene from pCHAmNF-1X2 (described below) into the multicloning site (between the NotI and XbaI restriction sites) of the pcDNA3 vector (Invitrogen). pCHAmNF1-X2 was a generous gift from Richard Gronostajski (The State University of New York at Buffalo) which contains the cDNA coding region of murine NF-1X (with a single amino acid difference, murine NF-1X is >99.7% homologous to human NF-1X). The NF-1X cDNA was subcloned into the pCHA vector to form a fusion protein with an N-terminal hemagglutinin tag. This tag has shown no effect on NF-1X DNA-binding or transactivation functions (6). pCHAmNF-1A1.1 (also from Richard Gronostajski at The State University of New York at Buffalo) is similar to pCHAmNF-1X2, described above, except that it contains the NF-1A1.1 gene rather than the NF-1X2 gene. pM1TC (9) is the entire JCV prototype, Mad-1 genome cloned into the EcoRI restriction site of pBR322.

Expression and purification of recombinant GST and GST-Jun proteins.

GST and GST-c-Jun were expressed in competent DH5-α Escherichia coli (Invitrogen) that was transformed with pGEX2T or pGEX-Jun, respectively.

GST pull-down assay.

GST and GST-c-Jun were generated, purified, and immobilized on glutathione-agarose as described above. Cultures of PDA were transiently transfected with pCDNA3NF-1X (2 μg/2 × 10⁶ cells) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. To generate whole-cell extracts, the PDA were disrupted 36 h posttransfection with ice-cold lysis buffer E (20 mM HEPES [pH 7.5], 150 mM NaCl, 3% glycerol, 5 mM MgCl₂, 1 mM CaCl₂, 1 mM EDTA, 1.0% NP-40, and one Complete Mini protease/phosphatase inhibitor tablet [Roche] per 20 ml). Lysates were sonicated on ice three times, 5 s each, and incubated separately with immobilized GST or GST-c-Jun at 4°C, rotating for 2 h. Resulting complexes were washed four times with 100-bed volumes of binding buffer containing 0.1% NP-40 and once with PBS. Bound proteins were eluted by boiling the complexes for 3 min in 2× PLB, resolved on 12% precast SDS-polyacrylamide ReadyGels (Bio-Rad), transblotted to PVDF membranes (Millipore), and analyzed by anti-NFX-1 (Geneka/Active Motif) chemiluminescent Western blotting.

In vitro binding assay.

GST and GST-c-Jun were generated and purified as described above. Employing pCDNA3NF-1X as a template, [³⁵S]methionine-labeled NF-1X ([³⁵S]NF-1X) was generated using the TNT Coupled in vitro transcription/translation system (Promega) according to the manufacturer's protocol. Approximately 2 μg of GST or GST-c-JUN immobilized on glutathione-agarose was incubated with [³⁵S]NF-1X (to form bead-complexes) in binding buffer (as described in the “Expression” section above) containing 1.0% NP-40 and 1 mg/ml bovine serum albumin. After incubation for 2 h at 4°C with agitation, the bead complexes were washed once in fresh binding buffer as used for the incubation above, twice in binding buffer containing only 0.1% NP-40, twice in binding buffer alone, and once in PBS. The washed bead complexes were disrupted by boiling for 3 min in Laemmli PLB and the components resolved by electrophoresis on 12% precast SDS-polyacrylamide ReadyGels (Bio-Rad). These gels were fixed and stained with Coomassie brilliant blue R-250 in 10% methanol and then dried and exposed to BioMax MR film (Kodak) overnight at −80°C.

PDA nuclear extract.

PDA (5 × 10⁶ cells/162-cm² flask) were washed with ice-cold Tris-buffered saline (TBS), scraped, pooled, and pelleted by centrifugation at 1,500 × g for 5 min at 4°C. Pellets from each flask were washed in 1 ml ice-cold TBS and resuspended in 400 μl of ice-cold NEB-A (nuclear extract buffer-A) (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and one Complete Mini protease/phosphatase inhibitor tablet [Roche] per 20 ml) and incubated for 15 min at 4°C to swell the cells. Twenty-five microliters of 10% NP-40 was added to the mixture followed by a brief vortex. The homogenates were centrifuged at 16,000 × g for 30 s at 4°C. The supernatants, containing cytosolic proteins, were collected and stored at −80°C. The nuclear pellets were resuspended in 50 μl ice-cold NEB-C (nuclear extract buffer-C) (20 mM HEPES, pH 7.9, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and one Complete Mini protease/phosphatase inhibitor tablet [Roche] per 20 ml). Samples were rocked for 15 min at 4°C and then centrifuged at 16,000 × g for 5 min at 4°C. Supernatants containing nuclear proteins were stored at −80°C. Protein concentrations were determined by using a Bio-Rad DC protein assay kit according to the manufacturer's protocol. Extracts were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on 4 to 12% gradient bis-Tris gels (Invitrogen), transferred to a PVDF membrane (Millipore), and analyzed by Western blotting as described in the “IP and Western blot” section but with anti-NF-1A, anti-NF-1B, and anti-NF-1X (Geneka/Active Motif), as well as anti-β-actin (Sigma) and anti-GFAP (Chemicon) primaries. In cases where monoclonal primary antibodies were used, a horseradish peroxidase-conjugated antimouse (Santa Cruz Biotechnology) secondary antibody was employed for detection.

JCV infection in PDA.

PDA were exposed to JCV, Mad-4 variant, at 200 hemagglutination units/10⁶ cells in a minimal covering of serum-free astrocyte medium. Ninety minutes after JCV exposure, the medium was supplemented with fresh complete astrocyte medium. Nuclear and cytosolic fractions of infected PDA were prepared 4 days post-JCV exposure, resolved by SDS-PAGE, and assayed by Western blotting as described in the “PDA nuclear extract” section but with anti-T antigen (Oncogene), a polyclonal rabbit anti-Vp-1 developed in our lab, and anti-β-actin (Sigma).

Overexpression experiments.

PDA were transfected with pCDNA3, pCDNA3NF-1X, or pET-c-Jun (a generous gift from James A. Goodrich, University of Colorado) using 5 μg of DNA/10⁷ cells with Amaxa (Amaxa Inc.) astrocyte nucleofector reagent. At 12 h posttransfection, the media were replaced with fresh medium containing JCV at 200 hemagglutination units/10⁶ cells. After 12 h, medium containing virus was aspirated, cells were washed once, and fresh medium was added. Nuclear fractions of transfected/infected PDA were prepared 4 days post-JCV exposure, resolved by SDS-PAGE, and analyzed by Western blotting as described in the “PDA nuclear extract” section but with anti-NF-1X (Geneka/Active Motif), anti-c-Jun (Santa Cruz Biotechnology), the polyclonal rabbit anti-Vp-1 developed in our lab, and anti-β-actin (Sigma).

ATP binding assay.

The Mad-1 JCV promoter was PCR amplified from pM1TC (9) using a modified forward primer (JCV Mad-1) (nt 4992 to 5011) having a biotin group linked to the 5-prime end and a normal reverse primer (JCV Mad-1) (nt 447 to 427). This biotinylated PCR product was coupled to streptavidin agarose beads, as previously described (30), to generate ATP-Mad-1. For preclearing of PDA extracts prior to ATP-Mad-1 exposure, an approximately 1,500-bp segment of NF-1A1.1 coding sequence (locus_ID: NM_010905) was PCR amplified from pCHAmNF-1A1.1 using a modified forward primer (nt 1 to 27) having a biotin group linked to the 5-prime end and a normal reverse primer (nt 1520 to 1500). This PCR product was coupled to streptavidin agarose beads to generate an anchored coding region (ACR-NF-1A1.1).

In an approach similar to antibody-based immunoprecipitations, ATP-Mad-1 slurry was used to pull down DNA binding proteins from aliquots of PDA nuclear extracts (same extracts as those described in “Overexpression experiments”). Briefly, PDA nuclear extracts were precleared by addition to equal volumes of protein binding buffer (1× TBS, pH 7.4, 1% Triton X-100, 1% glycerol, and protease inhibitors) containing 10% ACR-NF-1A1.1 and gently rotated overnight at 4°C. After centrifugation to pellet ACR-NF-1A1.1, the supernatants (precleared nuclear extracts) were added to equal volumes of protein binding buffer containing 10% ATP-Mad-1. After 2 h, at 4°C with gentle rotation, the ATP-Mad-1 was pelleted by centrifugation and washed three times with ATP wash buffer (1× TBS, pH 7.4, 0.5% Triton X-100, 0.5% glycerol, and protease inhibitors) and once with TBS containing protease inhibitors. Bound proteins were eluted by boiling the washed ATP-Mad-1 for 3 min in Laemmli PLB, resolved by SDS-PAGE, and analyzed by Western blotting as described in the “PDA nuclear extract” section but with anti-NF-1X (Geneka/Active Motif) and anti-c-Jun (Santa Cruz Biotechnology).

Inhibition of NF-1 binding by rhAP-1(c-Jun).

We previously identified the juxtaposition of the NF-1 and c-Jun binding sites in JCV promoters using DNase footprinting analysis (1, 2) and have posed the hypothesis that there is direct protein-protein interaction between NF-1 and c-Jun. To investigate this hypothesis and the effect c-Jun may have on NF-1 binding to a JCV promoter, EMSA assays were performed using a radiolabeled oligonucleotide probe containing the NF-1 consensus site derived from the JCV Mad-1 sequence (NF-1 probe) (Fig. 1). A gel-shifted band specific for binding of NF-1 proteins was observed when stromal cell extract was incubated with the NF-1 probe (Fig. 1, lane 3). The presence of a molar excess of unlabeled NF-1 probe (cold) competed out binding to the NF-1 probe (Fig. 1, lane 4), while an excess of an altered unlabeled probe containing a mutated NF-1 consensus site (mutant) did not reduce binding, demonstrating specificity (Fig. 1, lane 5). A primary 30-min incubation of stromal cell extract with the NF-1 probe, followed by a secondary 30-min incubation after the addition of rhAP-1(c-Jun), reduced binding to the NF-1 probe to some extent (Fig. 1, lane 6). However, reversing the order by first incubating with rhAP-1(c-Jun) and then adding stromal cell nuclear extracts eliminated NF-1 protein binding to the consensus sequence altogether, suggesting a significant inhibitory interaction (Fig. 1, lane 7). This inhibition of NF-1 binding through primary c-Jun incubation was dose dependent. Reducing the concentration of rhAP-1(c-Jun) by 4-, 16-, or 64-fold decreased the inhibitory effect proportionally (Fig. 1, lanes 8 to 10). As a negative control, NF-κB, which like rhAP-1(c-Jun) is not recognized by the NF-1 probe, had no affect on binding to the NF-1 probe (Fig. 1, lanes 11 and 12). These results demonstrate that c-Jun can inhibit binding of the NF-1 protein to the consensus NF-1 site of the JCV Mad-1 promoter if introduced prior to and/or in sufficient quantity with an NF-1 source (e.g., stromal cell extract). Interestingly, the inhibition of NF-1 binding occurs independently of c-Jun binding the NF-1 probe, as demonstrated by the failure of rhAP-1(c-Jun) to shift the NF-1 probe by itself (Fig. 1, lane 2). Free NF-1 probe was routinely run off the bottom the EMSA gels to avoid interference with the clear identification of entire NF-1 smears. In EMSAs where the NF-1 probe was not run off the bottom of the gel (data not shown), addition of rhAP-1 showed no reduction in the free NF-1 probe band compared with lanes having no rhAP-1, ruling out rhAP-1-mediated isotope cleavage from the probe. Therefore, the observed inhibition may be the result of a physical interaction between NF-1 and c-Jun that prevents NF-1 from binding to the JCV promoter and/or an effect of free c-Jun on the NF-1 consensus site, such as electrostatic interactions that have been described by others (27, 28).

IP and Western blot analysis for NF-1 and c-Jun.

To identify specific interactions between NF-1 class members and c-Jun, we first examined whether these proteins physically bind each other. Western blot analyses were conducted with stromal and PDA total cell lysates subjected to immunoprecipitation (IP) with either anti-NF-1X or an antirabbit immunoglobulin G (IgG) control and then resolved by PAGE and probed with anti-c-Jun. As shown in Fig. 2, c -Jun coprecipitated with NF-1X in both stromal and astrocyte lysates (Fig. 2A, lanes 1 and 3). No binding from the cell lysates was seen with the antirabbit IgG control (Fig. 2A, lanes 2 and 4). These data demonstrate that NF-1 and c-Jun were physically associated in the lysates of both stromal cells and PDA. The detection of NF-1X in reciprocal co-IPs (anti-c-Jun precipitation, followed by anti-NF-1X Western blotting) was problematic, because NF-1X resolves near the 55-kDa size of IgG heavy chains.

GST pull-down assays.

GST pull-down assays were utilized in which recombinant GST and GST-c-Jun were separately incubated with the total cell lysates of either nontransfected PDA or pcDNA3NF-1X-transfected PDA (Fig. 2B). Proteins dissociated from the isolated (pulled-down) GST and GST-c-Jun were resolved on gels and analyzed in Western blots versus anti-NF-1X. While the constitutively expressed NF-1X in nontransfected PDA lysate showed an association with GST-c-Jun, the overexpression of NF-1X in the transfected PDA more clearly demonstrated this association. These experiments substantiate our findings of a direct binding interaction between NF-1X and c-Jun.

In vitro translation of NF-1X.

An additional GST pull-down assay was performed using the recombinant GST and GST-c-Jun described above, along with in vitro-transcribed/translated ³⁵S-labeled-NF-1X protein ([³⁵S]NF-1X) to rule out possible effects of other cellular proteins on the observed association of NF-1X and c-Jun (Fig. 2C). [³⁵S]NF-1X showed specific association with GST-c-Jun, further demonstrating the specific interaction between NF-1X and c-Jun.

Comparative NF-1 expression in progenitors and PDA.

To uncover additional details regarding the control of JCV activity by NF-1X, we analyzed NF-1 protein expression in our model cell culture system of multipotential human CNS progenitors. Progenitors support only low levels of JCV activity compared with that for PDA (23). Our studies here revealed differential protein expression patterns of NF-1 class members, with elevated expression of NF-1X in PDA (Fig. 3A). β-Actin detection served as the control for equivalent total protein loading between progenitor and PDA nuclear extracts. The cytosolic fraction probed with anti-GFAP (a cytoskeletal marker that specifically identifies astrocytes among other CNS cell types) was included to illustrate the complete differentiation from progenitor to PDA.

JCV infections in the multipotential human CNS progenitor-cell culture system.

JCV activity in human CNS progenitors and PDA was analyzed at 4 days post-JCV exposure (the time interval used for all JCV infection studies presented here) by Western blot assays of nuclear extracts (Fig. 3B). At this time point, PDA showed significant expression of JCV early (T) and late (Vp-1) proteins compared with levels for progenitor cells. In subsequent experiments, levels of Vp-1 expression were used as the measure of JCV activity. This experiment demonstrates the positive role of NF-1X on JCV activity.

Interference of c-Jun on NF-1 binding to JCV promoter.

Since some AP-1 binding sites are found adjacent to NF-1 binding sites in JCV promoters, we explored how increased protein expression of either c-Jun or NF-1X might affect protein interactions with JCV promoter sequence (specifically Mad-1) and if interference with NF-1 binding alters JC viral activity. We used ATP binding experiments to compare the binding levels of NF-1X and c-Jun from nuclear extracts of JCV-infected PDA transfected to overexpress NF-1X or c-Jun. Proteins that bound ATP-Mad-1 were eluted and analyzed by Western blotting using anti-NF-1X or anti-c-Jun. PDA transfected to overexpress NF-1X showed a corresponding increase in NF-1X association to ATP-Mad-1 and an increased Vp-1 level compared with control PDA (Fig. 4). On the other hand, the increased association of c-Jun with ATP-Mad-1 in PDA transfected to overexpress c-Jun was partnered with a decrease in Vp-1 expression. This finding suggests that increased c-Jun association with AP-1 binding sites could be responsible for interference with NF-1X binding to adjacent NF-1 sites. Therefore, the inability of NF-1X to bind efficiently to particular NF-1 consensus binding sites in JCV promoters as a result of c-Jun interaction is, in the same measure, playing a role in the observed decrease of JCV activity (Fig. 4 and 5).