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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Virology. Author manuscript; available in PMC Apr 10, 2013.
Published in final edited form as:
PMCID: PMC3579630

Cooperative roles of NF-κB and NFAT4 in Polyomavirus JC regulation at the KB control element


The human polyomavirus JC (JCV) is the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is extremely common and after primary infection, JCV persists in a latent state. However, PML is a very rare disease suggesting that the virus is tightly regulated. Previously, we showed that NF-κB and C/EBPβ regulate the JCV early and late promoters via a DNA control element, KB, which also mediates the stimulatory effects of proinflammatory cytokines such as TNF-α on JCV gene expression. Other studies have implicated NFAT4 in JCV regulation. We now report that NFAT4 and NF-κB interact at the KB element to co-operatively activate both JCV early and late transcription and viral DNA replication. This interplay is inhibited by C/EBPβ and by agents that block the calcineurin/NFAT signaling pathway. The importance of these events in the regulation of JCV latency and reactivation is discussed.

Keywords: Polyomavirus JC, NFAT signaling, NF-kappaB signaling, progressive multifocal leukoencephalopathy


The human neurotropic polyomavirus JC (JCV) is the causative agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML) (Padgett et al., 1971). PML is caused by the reactivation of latent infection of JCV in the CNS and is usually characterized by multiple regions of demyelination, which are developed upon lytic infection of oligodendrocytes, the myelin-producing cells of the CNS, by JCV (Berger, 2007; Khalili et al., 2008). PML is a very rare disease even though the proven etiological agent is very common within the human population (White and Khalili, 2011). Most people are infected with JCV in childhood and this initial infection is not known to be related to any clinical disease. Subsequently, JCV remains in a persistent but dormant state known as latency, which is thought to be maintained due to the action of the immune system. Thus, PML occurs almost exclusively in individuals with impaired immune function. Once a very rare disease, PML has gained attention due to the mounting body of evidence showing reactivation of JCV in individuals with a variety of immunodeficiency disorders. These include persons with HIV-1/AIDS (Berger and Concha, 1995), patients with autoimmune disorders receiving immunotherapeutic drugs, e.g., Natalizumab (Kleinschmidt-DeMasters and Tyler, 2005; Langer-Gould et al., 2005; Van Assche et al., 2005; Khalili et al., 2007), Rituximab (Clifford et al., 2011), and Efalizumab (Kothary et al., 2011). PML can also occur in transplant recipients receiving immunosuppressive drugs to prevent rejection (Kumar, 2010; Mateen et al., 2011) and individuals with lymphoproliferative and myeloproliferative disorders (D’Souza et al., 2010) or who are chronically immunosuppressed due to a variety of other reasons (reviewed by Berger, 2007; Khalili et al., 2008). Many important aspects of PML pathogenesis remain unclear including the molecular basis and tissue site(s) of viral latency and the reactivation mechanisms that upregulate viral transcription and replication and induce PML (reviewed in Berger, 2011; White and Khalili, 2011). A variety of tissues have been reported to harbor latent JCV DNA including kidney, tonsils, bone marrow and indeed the brain itself (Elsner and Dorries, 1992; Greenlee et al., 2005; Mori et al., 1991, 1992; Perez-Liz et al., 2008; Tan et al., 2010; White et al., 1992). The possibility that JCV is present in the brain prior to onset of overt disease suggests that the normal brain may be a site of viral latency and that JCV reactivation events may occur in situ and involve CNS factors such as cytokines that upregulate the expression of viral genes in glial cells, though the importance of entry of JCV into the brain as another barrier to PML should not be discounted (Berger, 2011).

The genome of JCV is a circular, closed, supercoiled DNA and is small in size (5,130 base pairs for the Mad-1 strain). It is composed of two regions, early and late, which are transcribed in opposite directions from a bidirectional promoter (Frisque et al., 1984; Imperiale and Major, 2007). This bidirectional promoter is also known as the noncoding control region (NCCR) and governs viral early and late genes in opposite directions of the circular polyomavirus DNA genome. The NCCR contains the binding sites for many transcription factors that regulate JCV gene expression. Signal transduction pathways that lie downstream of extracellular growth factors and immunomodulators such as proinflammatory cytokines, e.g., TNF-α, IL-1β, IL-6, regulate some of these transcription factors. Our earlier work indicated the involvement of the NF-κB signaling pathway in the activation of JCV transcription (Mayreddy et al., 1996; Ranganathan and Khalili, 1993; Romagnoli et al., 2009; Safak et al., 1999; Wollebo et al., 2011). The unique site for NF-κB has been designated the KB element and is located in the NCCR on the early side of the origin of viral DNA replication. The KB element has shown to be a functional NF-κB binding site by gel shift studies with the NF-κB p65 subunit and activates JCV gene expression in response to PMA (Ranganathan and Khalili, 1993). Our earlier observations on JCV transcription indicate that the KB element is positively regulated by NF-κB p65 binding and negatively regulated by isoforms of the C/EBPβ protein, which bind to an adjacent site within the KB element (Romagnoli et al., 2009). In these experiments, a ternary complex of NF-κB/p65, C/EBPβ-LIP and JCV KB DNA could be detected and mutagenesis analysis indicated that the KB element regulates both basal and p65-stimulated transcription. We have also found that TNF-α stimulated both early and late JCV transcription through the KB element and that KB was able to confer TNF-α responsiveness to a heterologous promoter (Wollebo et al., 2011). Interestingly, Manley et al. (2006) reported that nuclear factor of activated T cells 4 (NFAT4) has a role in JCV infection of glial cells and suggested that it binds to the same region (KB element).

NFAT transcription factors were first described in lymphocytes and remain one of the best-characterized targets for dephosphorylation by calcineurin, a cell signaling phosphatase involved in T cell activation (Feske et al., 2003). Many different roles for NFAT have now been described in non-lymphoid tissue including neurons and glia (Graef et al., 1999, 2001; Ho et al., 1994; Mosieniak et al., 1998; Stevenson et al., 2001). There are currently five known members of the NFAT family including NFAT1 (NFATp), NFAT2 (NFATc), NFAT3 (NFATc4), NFAT4 (NFATc3/NFATx) and NFAT5 (Vihma et al al., 2008). NFAT4 is the only NFAT family member that is expressed in the astroglial cells, U-87 MG and SVGA (Manley et al., 2006). The first four members of the NFAT family are calcium regulated. The activity of the proteins is determined by their phosphorylation state, which in turn is tightly regulated by interplay between calcineurin and opposing kinases. Constitutively-expressed NFAT proteins reside in the cytoplasm. When calcineurin is activated through an increase in intracellular calcium level, NFAT is dephosphorylated at a large number of phosphorylated serine residues and rapidly enters the nucleus. NFAT nuclear accumulation is rapid and reversible (Shibasaki et al., 1996). Export occurs following rephosphorylation of NFAT by kinases, including GSK-3, most likely by remasking the NLS and allowing a constitutively active nuclear export signal to dominate (Beals et al., 1997).

In the context of our earlier work on the control of JCV transcription by the KB element, Manley et al (2006) reported that the immunosuppressive drug cyclosporin A, which inhibits calcineurin and activation of NFATS also inhibits JCV infection of glial cells and that glial cells express only NFAT4, which was found to bind to the JCV promoter in ChIP assays. Thus, it was of interest to examine the interplay of NF-κB and NFAT4 at the KB element of the JCV NCCR. We found that the transcription factors NFAT4 and NF-κB p65 can each stimulate both early and late gene expression activity through their binding to the KB element of the JCV noncoding control region when expressed alone. When NFAT and p65 are co-expressed, a cooperative stimulation of these activities was observed.


NFAT4 and NF-κB p65 cooperate to stimulate JCV early and late transcription via the KB element

In previous studies, we have identified an element (KB) in the JCV noncoding control region that binds NF-κB p65 and mediates the stimulation of JCV early and late transcription caused by NF-κB p65 expression and treatment of cells with PMA or TNF-α (Ranganathan and Khalili, 1993; Mayreddy et al., 1996; Safak et al., 1999; Romagnoli et al., 2009; Wollebo et al., 2011). Manley et al (2006) report that the transcription factor, NFAT4, which is the only NFAT family member to be expressed in U-87 MG and SVGA cells, may also act at this site to stimulate JCV transcription. Accordingly, it was of interest to investigate the effects of NF-κB p65 and NFAT4, each alone and in combination on early and late JCV transcription. As shown in Fig. 1A, transient transfection of increasing amounts of NFAT4 expression plasmid proportionately stimulated both early and late transcription measured using luciferase reporter plasmids. As we have reported before (Romagnoli et al., 2009), expression of p65 also stimulated JCV early transcription (Fig. 1B, lanes 1–5). When increasing amounts of p65 were expressed in the presence of NFAT4, there was a synergistic enhancement of JCV early transcription (Fig. 1B, lanes 6–8). A synergistic effect of p65 and NFAT4 was also observed for JCV late transcription (Fig. 1B, lanes 9–12). Recently, we described two KB element mutants (m1 and m2, Romagnoli et al., 2009) that are defective in basal early transcription and fail to respond to NF-κB p65 expression (Romagnoli et al., 2009) or to the stimulation of cells with PMA or TNF-α (Wollebo et al., 2011). These two mutants were tested for their response to NFAT4 expression (Fig. 1C). The two mutants had lower rates of basal transcription and did not respond to NFAT4 (Fig. 1C, lanes 3–6) while the wild-type promoter had a higher basal rate of transcription (lane 7) and was inducible by NFAT4 (lane 8). The activity of the basal wild-type JCV early CAT reporter promoter was about significantly higher (about four-fold) than either m1 or m2 and is marked with asterisk (Fig. 1C, lane 7). The activity of wild-type in the presence of NFAT was increased to a smaller extent and is marked with a double asterisk (Fig. 1C, lane 8) while this difference lower in extent to that seen with the wild-type JCV early LUC reporter promoter (Fig. 1A), it was significant and reproducible. In a previous study (Wollebo et al., 2011), we showed that the KB element was able to confer, upon a heterologous constitutive promoter (Herpes simplex virus thymidine kinase - tk), the ability to respond to TNF-α and the transient transfection of expression plasmid for the transcription factor NF-κB p65. NFAT4 is known to be activated by the calcium-dependent phosphatase calcineurin (Clipstone and Crabtree, 1992). As shown in Fig. 1D, tk reporter plasmid containing the JCV wild-type KB element was activated by the calcium ionophore ionomycin as well as by PMA (wt-tk, lanes 9–12). No activation was seen for the native tk promoter (lanes 1–4) or with a promoter containing a mutant KB element (mt-tk, lanes 5–8).

Figure 1
Effect of ectopic expression of NFAT4 and NF-κB on JCV early and late promoter reporter expression

C/EBPβ LIP isoform inhibits basal, NFAT4-stimulated and NFAT4/NF-κB p65-stimulated transcription for both the JCV early and late promoters

In addition to the stimulatory effect of NF-κB p65 on the KB element, we previously found that the transcription factor C/EBPβ binds to the same site and inhibits basal and NF-κB-stimulated JCV transcription (Romagnoli et al., 2009). Of the three C/EBPβ isoforms (full-length, LIP and LAP), C/EBPβ LIP was the strongest in this regard and was shown to bind to the KB element by gel shift and ChIP analyses and co-immunoprecipitation with antibody to NF-κB p65 (Romagnoli et al., 2009). Thus it was of interest to investigate the effects of C/EBPβ LIP and NFAT4, each alone and in combination on JCV early and late transcription. As shown in Fig. 2A, C/EBPβ LIP inhibited basal and NFAT4-stimulated transcription of both the JCV early and late promoters. In addition, as shown in Fig. 2B, if NFAT4 and p65 were coexpressed, their cooperative effects on both early and late transcription (lanes 3 and 7 respectively) were strongly inhibited by C/EBPβ LIP (lanes 4 and 8). Thus C/EBPβ LIP inhibits basal, NFAT4-stimulated and NFAT4/p65-stimulated JCV early and late transcription.

Figure 2
Effect of ectopic expression of NFAT4 and C/EBPβ on JCV early and late promoter reporter expression

Cyclosporine inhibits JCV replication

The immunosuppressive drug cyclosporine A is an inhibitor of the calcium-calmodulin-dependent protein serine phosphatase calcineurin (Clipstone and Crabtree, 1992), which dephosphorylate and thereby activate NFATs (Li et al., 2011). Manley et al (2006) reported that cyclosporine reduces efficiency of JCV infection consistent for a role of NFATs in the JCV life cycle. Accordingly, we transfected/infected SVGA cells with JCV in the presence and absence of cyclosporine and measured viral DNA replication using the DpnI assay (Fig. 3). DpnI digests transfected input DNA because it is bacterial in origin and contains methylated adenine residues but DpnI does not digest DNA that has been replicated. As shown in Fig. 3A, a band of replicated DNA corresponding in size to the plasmid restriction fragment of JCV genomic DNA (lane 1) was observed in the infected cells, untreated and cyclosporine-treated (lanes 3 and 4 respectively). Comparison of the intensities of the bands measured using Bio-Rad QuantityOne software indicated that cyclosporine caused an 80% inhibition of JCV DNA replication. From the same infection experiment, we also harvested protein samples and performed Western blots for the JCV late proteins VP1 and agnoprotein (Fig. 3B). Cyclosporine caused a 50% and 40% reduction in the level of VP1 and agnoprotein expression respectively (compare lanes 3 and 4).

Figure 3
Effect of cyclosporine on JCV replication

JCV early and late transcription are strongly inhibited by VIVIT

In order to identify more potent and selective inhibitors of NFAT activation than cyclosporine, Aramburu et al (1999) employed an affinity-driven peptide selection approach to select a high-affinity calcineurin-binding peptide from combinatorial peptide libraries based on the calcineurin docking motif of NFAT. An optimal peptide MAGPHPVIVITGPHEE (referred to as VIVIT) was identified which, when expressed as a fusion protein with green fluorescent protein (GFP), efficiently inhibited calcineurin-dependent nuclear translocation of NFAT (Aramburu et al., 1999). To test the effect of VIVIT inhibition of NFAT on JCV early transcription, we transfected U-87 MG cells with JCV early reporter plasmid and various combinations of expression plasmids for NFAT4, p65 and GFP-VIVIT (Fig. 4A). VIVIT strongly inhibited basal and NFAT4-stimulated early promoter activity (compare lane 1 to lane 3 and lane 2 to lane 4). Interestingly, VIVIT also inhibited p65-stimulated early promoter activity (compare lane 5 to lane 6). If GFP replaced GFP-VIVIT, no effect on transcription was observed (data not shown). When the experiment was performed with the JCV late promoter, similar results were obtained (Fig. 4B). In the next series of experiment, we examined the effect of VIVIT in the presence of JCV T-antigen on JCV early (Fig. 4C) and late transcription (Fig. 4D). In both cases, VIVIT strongly inhibited transcription. Remarkably, the late promoter was stimulated over thirty-fold by T-antigen (Fig. 4D, compare lanes 1 and 5) but this was reduced to only two-fold in the presence of VIVIT (lane 6).

Figure 4
Effect of ectopic expression of VIVIT on NFAT4 and NF-κB stimulation of JCV early and late promoter reporter expression

Both NFAT4 and p65 bind to KB element DNA in gel shift assays

Our previous work had characterized the binding of NF-κB p65 and C/EBPβ LIP to the KB element of the JCV NCCR using gel shift assays (Romagnoli et al., 2009). To characterize NFAT4 binding, we performed a series of gel shift experiments with the KB element probe and nuclear extracts from cells expressing combinations of NFAT4, p65 and C/EBPβ LIP (Fig. 5A). Since, we were unable to produce a supershift after trying several different antibodies to NFAT4, we included a series of gel shifts in the presence of GFP-VIVIT to allow the identification of the NFAT4 gel shift band (Fig. 5A, lower panel). When either NFAT4 (lane 3) or p65 (lane 9) were expressed alone, no extra band shift was observed compared to the control (lane 2). However, co-expression of NFAT4 and p65 (lane 4), produced a new band migrating at a higher shift (band position 3). Further, this band was eliminated in the presence of VIVIT (lane 14, band position 3) indicating that NFAT4 is present in this complex. Similarly, co-expression of p65 and LIP also produced a new band migrating at a higher shift (band position 3) since these two proteins are known to form a ternary complex (Romagnoli et al., 2009). In contrast, this complex was not eliminated in the presence of VIVIT (lane 16, band position 3) as expected. The presence of p65 in band position 2 was confirmed by supershift with antibody to p65, which supershifted it to band position 1, which is close to the wells (lane 10). Cold competitor KB element DNA eliminated all the bands (lanes 8 and 18).

Figure 5
Gel shift analysis of transcription factor binding to the JCV KB element

VIVIT inhibits JCV replication

Next, we used VIVIT to investigate a role for NFAT in viral replication using DpnI assay and Southern blot as described above. Accordingly, we transfected/infected SVGA cells with JCV in the presence and absence of co-transfection of GFP-VIVIT or GFP expression plasmids and measured viral DNA replication using the DpnI assay (Fig. 6). As shown in Fig. 6A, a band of replicated DNA corresponding in size to the plasmid restriction fragment of JCV genomic DNA (lane 1) was observed in the infected cells, GFP and GFP-VIVIT-transfected (lanes 4 and 5 respectively). Comparison of the intensities of the bands measured using Bio-Rad QuantityOne software indicated that VIVIT caused a 60% inhibition of JCV DNA replication. Similarly, in the same infection experiment, Western blots for the JCV VP1 and agnoprotein showed that VIVIT caused a 60% and 80% reduction in the levels of VP1 and agnoprotein expression respectively (Fig. 6B).

Figure 6
Effect of VIVIT on JCV replication


Our data indicate that the transcription factors NFAT4 and NF-κB p65 stimulate both early and late gene expression activity through their binding to the KB element of the JCV noncoding control region. When NFAT and p65 are co-expressed, a co-operative stimulation of these activities was observed. C/EBPβ, which can bind to the KB element in complex with NF-κB to inhibit early and late transcription (Romagnoli et al., 2009), also inhibited the transcription stimulation of NFAT4 and combined NFAT4/NF-κB. Similarly, the NFAT inhibitor VIVIT inhibits not only early and late transcription stimulated by NFAT4 but also by p65. These data suggest a co-operative interplay between NFAT4 and NF-κB and this was supported by a novel gel shift band that was detected when these factors were co-expressed. Thus NFAT4 and NF-κB, the effectors of two different signal transduction pathways that are regulated by extracellular stimuli, converge at a common point on the JCV KB control element, which may be an important regulatory event for the status of viral activation. There have been other reports of promoter/enhancer elements that are regulated cooperatively by NFAT and NF-κB proteins including the NF-κB site localized in intron 1 of the ICAM-1 gene (Xue et al., 2009) and the C3-3P NF-κB site of the IFN-γ promoter (Sica et al., 1997).

What is the importance of this interaction in viral regulation? The life cycle of JCV is marked by an ability to exist in a latent state, where neither transcription nor replication occur, and then to reactivate leading host cell lysis and PML. Detection of latent JCV DNA has been reported in a variety of different tissues (reviewed in Berger, 2011; White and Khalili, 2011). Importantly, the brain itself has been reported to harbor latent virus (Elsner and Dorries, 1992; Greenlee et al., 2005; Mori et al., 1991, 1992; Perez-Liz et al., 2008; Tan et al., 2010; White et al., 1992). Of note, the virus found in the brain has a rearranged NCCR region that is characteristic of pathogenic forms of the virus, also known as “PML-type” and not the archetypal form of the virus that is found in the kidney (Perez-Liz et al., 2008; Tan et al., 2010; White et al., 1992). The rearrangements in the late-proximal region of the JCV NCCR are undoubtedly important in pathogenesis since they allow the duplication of important promoter/enhancer elements, as typified by the 98 base-pair repeat of the Mad-1 strain, and also the deletion of archetypal elements that may restrict promoter activity (Daniel et al., 1996). However, the finding of PML-type viral DNA that is not undergoing transcription or replication in astrocytes and oligodendrocytes from normal brain implies that mechanisms must exist for switching the JCV NCCR “on and off”.

We hypothesize that the KB element may provide the basis for such a switching mechanism. Previously, we had found that the highly conserved early proximal portion of the JCV NCCR contains an element, KB, which binds and is activated by NF-κB (Ranganathan and Khalili, 1993; Mayreddy et al., 1996; Safak et al., 1999; Romagnoli et al., 2009) and confers the ability to be activated by TNF-α on both the JCV early and late promoters (Wollebo et al., 2011). In another study, Manley et al (2006) reported that NFAT4 was required for JC virus infection. We have now confirmed and extended these observations by demonstrating that NF-κB and NFAT4 co-operatively activate the transcriptional status of JCV through the KB element. Since a majority of cases of PML occur in the context of HIV-1/AIDS and HIV-1 can generate cytokine cascades in the CNS involving proinflammatory cytokines including TNF-α (Benveniste, 1994; Kaul et al., 2005; Wesselingh et al., 1993; Yeung et al., 1995), and we have described a role for TNF-α acting through the KB site in the stimulation of JCV transcription (Wollebo et al., 2011), it is possible that JCV reactivation in PML involves co-operative interaction between NF-κB and NFAT pathways downstream of TNF-α and converging at the KB element. This is supported by the presence of robust levels of TNF-α and TNFR1 in PML lesions in HIV/PML clinical samples (Wollebo et al., 2011). As well as the well-established signaling by TNF-α through NF-κB, it has been reported that TNF-α can activate NFAT signaling (Yarlina et al., 2008).

In an alternative scenario, another cytokine such as IL-1β, which evokes NFAT activity in primary astrocytes (Furman et al., 2010; Sama et al., 2009) and is also upregulated in the CNS in HIV/AIDS “cytokine storms” (Benveniste, 1994; Kaul et al., 2005) may be involved in activation of the NFAT pathway and may act synergistically with TNF-α/NF-κB signaling to activate JCV through the KB element. IL-6 is another cytokine that has been reported to be upregulated in the CNS in HIV/AIDS (Benveniste, 1994; Yeung et al., 1995) and can activate NFAT signaling (Diehl and Rincón, 2002).

It is also possible in HIV/PML that HIV itself might impart a direct effect on the JCV KB element through the action of the HIV transactivator protein Tat, which is well established as an activator of JCV (Tada et al., 1990). HIV Tat has the ability to increase the activity of NF-κB (Mahlknecht et al., 2008; Taylor et al., 1992) and can also stimulate signaling through the NFAT pathway (López-Huertas et al., 2010; Pessler and Cron, 2004; Vacca et al., 1994). It should be noted that HIV-1 provirus itself is also regulated by NF-κB, NFAT and Tat and that these signaling proteins are thought to be involved in the activation of latent HIV-1 transcription when resting T-cells become activated (Coiras et al., 2009; Kinoshita et al., 1997; Pessler and Cron, 2004).

In summary, these studies suggest that the inactive latent JC virus that has been detected in the normal brain of some individuals may be subject to a switching mechanism via the KB control element whereby cooperative actions involving the NF-κB and NFAT signaling pathways (and perhaps the downregulation of the inhibitory effect of C/EBPβ), which may promote JCV reactivation that are transduced from such cytokines as TNF-α, and perhaps others, acting directly on glial cells. Thus, cytokine storms in the CNS and perhaps direct effects of HIV-1 Tat could initially activate JCV transcription, followed by the onset of viral replication and the spread of virus in the context of impaired immunosurveillance, allowing the formation of PML lesions. A deeper understanding of these processes may suggest new ways to prevent and treat PML.


Two transcription factors, NF-κB p65 and NFAT4, which lie downstream of different signal transduction pathways, have the capacity to bind to a common element (KB) in the JCV noncoding control region and thereby cooperatively activate both early and late transcription. Inhibition of either NFAT4 by calcineurin inhibitors such as cyclosporine or VIVIT or NF-κB, by C/EBPβ LIP, prevents this activation. This novel mechanism may be involved in the reactivation of JCV from latency in glial cells in response to extracellular cytokines.


Cell culture and plasmids

U-87 MG human glioblastoma and SVGA were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Reporter constructs, JCVE-LUC and JCVL-LUC contained the JCV promoter from the Mad-1 strain linked to the luciferase gene in the early and late orientations respectively (Wollebo et al., 2011). The promoter reporter constructs, JCVE-CAT and JCVL-CAT contained the JCV promoter from the Mad-1 strain linked to the chloramphenicol acetyltransferase (CAT) gene in the early and late orientations respectively (Chen and Khalili, 1995). JCVE-CAT promoter mutants m1 and m2 were made by site-directed mutagenesis at two adjacent sites within the KB site as we previously described (Romagnoli et al., 2009). Heterologous reporter plasmids, pBLCAT2-wt-kB and pBLCAT2-mt-kB were described previously and were made by cloning tandem wild-type or mutant JCV κB sites respectively into the CAT reporter plasmid pBLCAT2, which contains the constitutive Herpes simplex virus thymidine kinase (tk) promoter (Wollebo et al., 2011). The expression plasmids pCMV-p65 and pCMV-LIP were described previously (Romagnoli et al., 2009). The pGFP-VIVIT construct expresses the NFAT peptide inhibitor VIVIT fused with green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter (Addgene, Cambridge MA). Plasmid containing the full-length human cDNA of NFAT4 was purchased from Open Biosystems (Huntsville, AL). The full length NFAT4 was PCR amplified from the plasmid pOTB7 and cloned in EcoR1 and Xho1 site of the pCDNA6A expression vector using the following primers. The forward primer was 5′-ctg gtt gaa ttc gcc acc atg act act gca aac tgt ggc-3′ and the reverse primer was 5′-cta aca ctc gag gag ccc atc aga tct tcc taa atc-3′.


Rabbit polyclonal anti-p65 (c-20, sc-372, Santa Cruz Biotechnology Inc., Santa Cruz, CA) and mouse monoclonal anti-C/EBPβ (H7, sc-7962, Santa Cruz) which recognizes all three C/EBPβ isoforms were used for Western blots. Mouse monoclonal anti-α-tubulin (clone B512) was from Sigma (St. Louis, MO) and mouse monoclonal anti-myc-Tag antibody (9B11) and rabbit polyclonal antibody to lamin A/C were from Cell Signaling Technologies, Inc. (Danvers MA). Mouse monoclonal antibody (Ab587) against JCV capsid protein VP1 was kindly provided by Dr. Walter Atwood, Brown University, Providence RI. Rabbit polyclonal antibody against JCV agnoprotein was previously described (Del Valle et al., 2002).

Western blots

Western blot assays were performed as previously described (White et al., 2006). Briefly, 50 μg of protein was resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary antibody (1/1000 dilution) and secondary antibody (1/10000 dilution). Bound antibody was detected with an ECL detection kit (Amersham, Arlington Heights, IL).

Transient transfection assays

Experiments involving co-transfection of reporter plasmids and expression plasmids were performed as we have previously described (Romagnoli et al., 2009; Wollebo et al., 2011). Briefly, U-87 MG cells were transfected with reporter constructs alone (5 μg) or in combination with the various expression plasmids. The total amount of transfected DNA was normalized with empty vector DNA. When PMA and ionomycin were used, 100 ng/ml PMA and 2 μM ionomycin were added to the cultures 90 min before harvesting. Assays for luciferase and CAT were performed as previously described (Romagnoli et al., 2009; Wollebo et al., 2011).

Gel shift assays

U-87 MG cells were transfected with and without the expression plasmids for 48 h and harvested. Nuclear proteins were then extracted and 10 μg were incubated with 50,000 cpm of a γ-32P-labeled double-stranded oligodeoxyribonucleotide probe as previously described (Romagnoli et al., 2009). The probe that was used in these gel shift experiments corresponded to the KB element: κB: 5′-aaaacaagggaatttccctggcctc-3′ (nts 5052-5078, JCV Mad-1 reference strain, GenBANK # NC_001699.


SVGA cells, which express SV40 T-antigen and support JCV replication were seeded at 1.5 × 106 cells/75 cm2 flask and transfected with wild-type Mad-1 genomic DNA using 7 μg with FuGene6 transfection reagent according to Manufacturer’s recommendations (Roche, Indianapolis, IN). In experiments to test the effect of VIVIT, cells were transfected with either 7 μg pGFP-VIVIT or pGFP (control) 24 hours before the viral DNA transfection. In experiments to test the effect of cyclosporine A, cells were treated 6 hrs before transfection/infection with cyclosporine A (10 μM final concentration). Cells were then maintained at 37°C in a humidified atmosphere with 7% CO2 for 10 days with every 3 days the medium changed for fresh medium with cyclosporine A until they were processed for protein extraction or DNA isolation. Protein extracts were analyzed by Western blot for VP1 and agnoprotein expression. After transfection/infection cells were harvested and low molecular weight DNA containing JC viral DNA was isolated using Qiagen spin columns as described by Ziegler et al., 2004). The JCV viral DNA was then digested with BamHI and DpnI restriction enzymes, resolved on 1% agarose gel and analyzed by Southern blotting using probe prepared from whole Mad-1 genome. DpnI is a methylation-sensitive of adenine residue enzyme that requires methylation of adenine, which only occurs in bacteria, and hence it digests transfected input DNA since but DNA that has been replicated. The intensity of the 5.13 Kb BamHI band in Southern blot is thus a measure of DNA replication.


We thank past and present members of the Center for Neurovirology for their insightful discussion and sharing of ideas and reagents. This work was supported by a grant awarded by the NIH to MKW.


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