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J Virol. May 2008; 82(9): 4250–4256.
Published online Feb 20, 2008. doi:  10.1128/JVI.02156-07
PMCID: PMC2293074

NF-κB-Mediated Activation of the Chemokine CCL22 by the Product of the Human Cytomegalovirus Gene UL144 Escapes Regulation by Viral IE86[down-pointing small open triangle]

Abstract

The product of the human cytomegalovirus (HCMV) gene UL144, expressed at early times postinfection, is located in the UL/b′ region of the viral genome and is related to members of the tumor necrosis factor receptor superfamily, but it does not bind tumor necrosis factor superfamily ligands. However, UL144 does activate NF-κB, resulting in NF-κB-mediated activation of the cellular chemokine CCL22. Consistent with this finding, isolates of HCMV lacking the UL/b′ region show no such activation of CCL22. Recently, it has been suggested that activation of NF-κB is repressed by the product of the viral gene IE86: IE86 appears to block NF-κB binding to DNA but not nuclear translocation of NF-κB. Intriguingly, IE86 is detectable throughout an infection with the virus, so how UL144 is able to activate NF-κB in the presence of continued IE86 expression is unclear. Here we show that although IE86 does repress the UL144-mediated activation of a synthetic NF-κB promoter, it is unable to block UL144-mediated activation of the CCL22 promoter, and this lack of responsiveness to IE86 appears to be regulated by binding of the CREB transcription factor.

Human cytomegalovirus (HCMV), a member of the Betaherpesvirinae subfamily, is one of the largest known double-stranded DNA viruses, with a genome of approximately 230 kb and containing at least 150 known open reading frames. It is widely distributed in human populations and generally causes a minor or asymptomatic infection after the primary infection of immunocompetent individuals. As with all herpesviruses, this infection persists for life. However, in the immunosuppressed or immunonaïve, the primary infection or reactivation can cause serious disease. There is no universally effective vaccine or antiviral treatment.

The ability of HCMV to repress or activate NF-κB at different times during infection remains a controversial subject. It is generally accepted that after the first few hours of infection, HCMV binding to the cell activates NF-κB (10, 23). However, we have shown that HCMV also downregulates tumor necrosis factor receptor 1 from the cell surface at early times of infection, which prevents NF-κB activation in response to external signaling (2). Similarly, there is evidence that at 72 h postinfection, some arms of the NF-κB-signaling pathway are also inhibited, rendering infected cells unable to respond to stimuli such as interleukin 1β (15). At intermediate times of infection (12 to 48 h), the NF-κB activation status of infected cells is unclear, although it has been suggested that prolonged activation of NF-κB by HCMV promotes viral replication and late gene expression (11). Consequently, it appears that NF-κB activation is important at certain stages of infection, and it would be counterproductive for the virus to inhibit NF-κB at these times.

UL144 is located in the UL/b′ region of the genome, which contains about 20 kb of DNA encoding at least 19 open reading frames but is dispensable for growth in vitro (8). It is related to members of the tumor necrosis factor receptor superfamily, is expressed at early times of infection, and negatively regulates TH1 cells by binding to the B- and T-cell lymphocyte activator BTLA (9). Additionally, like LMP-1 of Epstein-Barr virus, UL144 activates NF-κB at early times of infection and induces expression of the macrophage-derived chemokine (MDC), also termed CCL22 (22, 25).

Although the CCL22 promoter contains binding sites for several transcription factors, such as STATs and CREB, it is the NF-κB binding site which is critical for upregulation by UL144 (25). Activation of CCL22 is likely to complement the actions of UL144 binding to BTLA, as CCL22 is a TH2 chemoattractant which acts to subvert the TH1 immune response (22). Furthermore, CCL22 also acts as a chemoattractant for cells of the myeloid lineage that may aid viral spread and dissemination (35).

Immediately upon infection, HCMV expresses the viral immediate early gene products pIE86 and pIE72, which are under the control of the major immediate early enhancer/promoter. Together with pIE72, pIE86 is a potent transactivator of HCMV genes and is known to interact with numerous host cell proteins, including p53, PCAF, and CREB (4, 6, 18). It has been observed previously that IE86 is able to bind to CREB as well as to CREB binding proteins and to activate both viral and cellular promoters (18, 30). However, IE86 is also able to negatively regulate its own transcription by binding to a DNA site in the major immediate early enhancer/promoter, resulting in efficient autorepression at later stages of infection (3, 16, 34, 28). Recent evidence also suggests that a block of NF-κB activation at 6 h postinfection is due to IE86: IE86 inhibits binding of NF-κB to promoters (31). Although the levels of IE86 RNA expression are lower at later times, IE86 protein can be detected throughout an infection with the virus (1). Nevertheless, at intermediate times of infection there is a stimulation of NF-κB mediated by a gene within the UL/b′ region (5), and we have shown that UL144 also activates NF-κB (25). Consequently, the mechanism by which UL144 can upregulate NF-κB in the presence of IE86 is not clear.

We show here that although IE86 does indeed block the NF-κB-mediated activation of a synthetic NF-κB-responsive promoter by UL144, it does not block UL144-mediated activation of the CCL22 promoter. Furthermore, we show that the presence of the CREB binding site in the CCL22 promoter appears to be crucial for the ability of UL144 to upregulate CCL22 in the presence of IE86. This finding suggests that the ability of IE86 to inhibit NF-κB-mediated activation of promoters is dependent on the type of promoter and, in particular, the context of NF-κB binding sites with respect to binding sites for other transcription factors.

MATERIALS AND METHODS

Cells and viruses.

HFF cells were maintained in Eagle's minimal essential medium containing 10% fetal calf serum. Primary human MDM cells were generated as described previously (19). Briefly, peripheral blood mononuclear cells were isolated by density gradient centrifugation, using Ficoll-Hypaque (Nycomed Pharma AS). Primary monocytes were obtained by adherence for 1.5 h at 37°C in 5% CO2. Following adherence, the monocytes were cultured in Iscove's modified Dulbecco's medium (Gibco-BRL) supplemented with 15% horse serum (Sera-Lab), 15% fetal calf serum, 2 mM l-glutamine (Gibco-BRL), 100 U penicillin ml−1, 100 μg streptomycin ml−1, and 5 × 10−5 M hydrocortisone sodium succinate (Sigma) for 5 days at 37°C in 5% CO2. After 5 days, the cultures were stimulated with 10 ng phorbol myristate acetate ml−1 overnight to induce differentiation to HCMV-permissive macrophages. Infections with HCMV AD169 and Toledo have been described previously (21, 2).

Plasmids.

Reporter plasmids with the firefly luciferase gene under the control of the human beta interferon (IFN-β) promoter, pIFΔ(−125)lucter; the synthetic PRD multimer reporters p(PRDI/III)5tkΔ(−39)lucter and p(PRDII)5tkΔlucter; and the constitutive β-galactosidase reporter plasmid pJATlac were provided by S. Goodbourn (St. George's Hospital Medical School, University of London, United Kingdom) and have been described previously (25, 27). Plasmids containing the wild-type CCL22 promoter (WT pGL3-CCL22−722) or with deletions (pGL3-CCL22−553, pGL3-CCL22−476, pGL3-CCL22−289, pGL3-CCL22−114, and pGL3-CCL22−80) or mutations (at NF-κB binding sites 1 and 2, TT-AG and CC-AA, respectively) have been described previously (22). pGL3-CCL22 plasmids with mutated CREB binding sites 1 and 2 were generated with a GT-CA mutation at both sites. Expression of the UL144 gene from the pCDNA3 vector has been described previously (25), and the pCDNA3-IE86 construct used has been previously described (7).

Gene expression and protein analysis.

For reporter gene assays, plasmids were transfected into HFF cells with Lipofectamine 2000, using standard protocols with modifications described previously (25). Lysates were prepared and analyzed for luciferase and β-galactosidase activities (25, 27). For Western blotting, cells were harvested in Laemmli buffer and probed with rabbit or mouse monoclonal primary antibodies, followed by horseradish peroxidase-conjugated secondary anti-mouse or rabbit immunoglobulins (Ig) (Cell Signaling), and visualized with ECL reagent as described previously (25). IE86 was detected with a custom HCMV fluorescein isothiocyanate (FITC)-conjugated antibody without Evans blue (Chemicon International), UL144 with an anti-T7 Tag mouse monoclonal antibody (Novagen) or an anti-UL144 rat monoclonal antibody (a kind gift from C. Benedict, La Jolla Institute, La Jolla, CA), and actin with rabbit polyclonal antibody (Cell Signaling). DNA immunoprecipitations were carried out as described previously (20) with antibodies to the transcription factors NF-κB (p50/p65; Santa Cruz), CREB (Cell Signaling), and SP1 (Cell Signaling) or an IgG monoclonal control (Chemicon International), followed by PCR with primers to the pGL3 flanking regions (Promega), endogenous PACT (12), or endogenous CCL22 (5′-GAACAGTGGGGTTGGAGAGAAGGA-3′ and 5′-AGCCCAGGTGTCTGCAAAGGGACA-3′).

Immunofluorescence.

Infected cells were fixed and permeabilized for immunofluorescence confocal imaging as described previously (26, 27). IE72/86 was detected by using custom HCMV FITC-conjugated antibody without Evans blue (Chemicon International). p65 was probed by using primary antibody (Santa Cruz) followed by goat anti-rabbit antibody conjugated to tetramethyl rhodamine isocyanate (Sigma). Coverslips were mounted in Citifluor media and examined using a Nikon microscope, Image Pro WCIF ImageJ software, and TCS NF/SP confocal software. The images were detected using excitation wavelengths of 488 and 568.

CCL22-specific ELISA.

Macrophage cells were transfected using the Amaxa electroporation system in accordance with the Amaxa protocol. At 48 h posttransfection, the supernatants were harvested and assayed by CCL22 enzyme-linked immunosorbent assay (ELISA) in accordance with the manufacturer's instructions (RND Systems).

RESULTS

IE86 does not prevent relocation of NF-κB.

The mechanism by which UL144 activates NF-κB is through the induction of nuclear relocalization. As the exact mechanism by which IE86 inhibits NF-κB activation is not known, we tested the ability of UL-b′-negative and -positive viruses to mediate the nuclear relocalization of NF-κB at the times of infection when both UL144 and IE86 are expressed. Figure Figure11 shows that at 48 h postinfection, cells infected with the HCMV strain AD169, which lacks UL-b′ (and therefore UL144) show a cytoplasmic localization of the endogenous p65 subunit of NF-κB (Fig. (Fig.1,1, top left panel). In contrast, the HCMV strain Toledo, which contains UL-b′, shows nuclear localization of p65 (Fig. (Fig.1,1, bottom left panel), yet both of these viruses express IE86 in the nucleus (Fig. (Fig.1,1, middle panels). These results suggest that the UL-b′-containing virus causes nuclear relocalization of NF-κB at 48 h postinfection, consistent with the observations that UL144 alone is able to relocate NF-κB to the nucleus (25) and that this is not prevented by IE86. This is consistent with the suggestion that rather than targeting the stability or localization of NF-κB, IE86 acts on NF-κB at the NF-κB promoter interface to exert any inhibitory effects (30).

FIG. 1.
UL144-containing viruses induce NF-κB nuclear relocalization. HFF cells were infected with HCMV AD169 (a UL144-negative strain) or Toledo (a UL144-positive clinical isolate) for 48 h. Infection was detected with FITC-conjugated anti-IE72/86 (green), ...

IE86 blocks the activation of some, but not all, NF-κB-responsive promoters.

It has previously been shown that IE86 specifically blocks NF-κB binding to promoters in response to various cytokines, including tumor necrosis factor, and virus infection at early times postinfection (30). Our preliminary studies are consistent with these findings. We find that IE86 blocks the activation of the NF-κB-dependent IFN promoter and the NF-κB-only responsive promoter, PRDII, by double-stranded RNA (dsRNA). However, IE86 has no effect on the activation of interferon regulatory factor 3 (IRF3) by dsRNA (data not shown). Therefore, IE86 specifically blocks NF-κB- mediated transcription, but not IRF3 activation, in response to dsRNA.

However, we have also previously shown that UL144 causes activation of NF-κB-responsive promoters at times of infection when IE86 would be easily detectable. This finding is perplexing, as it is established that IE86 inhibits NF-κB binding to promoters. Therefore, we tested IE86 and UL144 in isolation and expressed together to determine if IE86 is able to prevent the activation of an NF-κB-only-driven promoter (PRDII) in response to UL144 in transfection assays. Figure Figure22 shows that IE86 does block the activation of PRDII in response to UL144 in that activation of the PRDII promoter by UL144 (Fig. (Fig.2A,2A, lanes 1 and 2) is prevented by IE86 (Fig. (Fig.2A,2A, lanes 3 and 4). Furthermore, this inhibitory effect of IE86 is titratable. Figure Figure2B,2B, lanes 1 to 5, shows that the activation of the PRDII promoter is blocked with increasing amounts of IE86. Consequently, at least for the PRDII promoter which is responsive to only NF-κB, NFκB-mediated activation is indeed prevented by IE86.

FIG. 2.
IE86 blocks UL144-induced activation of NF-κB in a dose-dependent manner. (A and B) HFF cells were transfected with p(PRDII)5tk-lucter and pJATlac and either a mammalian expression plasmid driving the overexpression of the HCMV protein IE86 or ...

However, UL144 also activates CCL22 expression in the context of viral infection via activation of NF-κB (25), and this occurs at times of infection when IE86 would still be detectable. Therefore, we next tested the ability of IE86 to prevent UL144-mediated expression from the CCL22 promoter, using transient transfection reporter assays and ELISA (Fig. (Fig.3).3). Clearly, UL144 activates the CCL22 promoter (Fig. (Fig.3A,3A, lanes 1 and 2) (25), and IE86 alone has a negligible stimulatory effect on the basal levels of activity of the CCL22 promoter (Fig. (Fig.3A,3A, lanes 1 and 3). However, when transfected together, the presence of IE86 has no inhibitory effect on the ability of UL144 to drive expression of luciferase from the CCL22 promoter (Fig. (Fig.3A,3A, lanes 3 and 4). Furthermore, IE86 does not prevent expression of endogenous CCL22 when induced by transfected UL144, as shown by ELISA (Fig. (Fig.3B),3B), even though substantial levels of IE86 are coexpressed (Fig. (Fig.3C).3C). Therefore, although IE86 does block UL144-mediated stimulation of an NF-κB-only-responsive promoter, it does not block UL144-mediated activation of the CCL22 promoter, even though this activation is also mediated by NF-κB.

FIG. 3.
IE86 does not prevent the induction of CCL22 by UL144. (A and B) HFF (A) or macrophage (B) cells were transfected with pGL3-CCL22 and pJATlac (A), expression plasmids for IE86 or UL144, or the control empty vector, as indicated by plus and minus signs. ...

The inability of IE86 to block UL144-mediated stimulation of the CCL22 promoter is due to the presence of a CREB-binding site.

To address the mechanism by which IE86 blocks NF-κB-mediated activation of a promoter bearing only an NF-κB site, but not the CCL22 promoter which contains other promoter binding sites in addition to NF-κB (Fig. (Fig.3),3), UL144 was tested for its ability to stimulate CCL22 promoter deletion mutants in the presence and absence of IE86. Activation of the CCL22 promoter by UL144 is not inhibited by the presence of IE86 (Fig. (Fig.33 and Fig. Fig.4A,4A, lanes 1 to 4). Sequential removal of −772 to −553 (Fig. (Fig.4A,4A, lanes 4 to 8), or −553 to −476 (Fig. (Fig.4A,4A, lanes 9 to 12) still resulted in the inability of IE86 to repress the CCL22 promoter. However, the removal of −476 to −209, which includes the removal of the CREB binding site, enabled IE86 to block the activation of the promoter by UL144 (Fig. (Fig.4A,4A, lanes 13 to 16, compare lanes 14 and 16). Neither of the two deletion mutant promoters (from nucleotides −1 to −114 or −1 to −60) which lack the NF-κB binding site was activated by UL144 (Fig. (Fig.4A,4A, lanes 17 to 24), consistent with previous findings (25). Therefore, the presence of the CREB binding site appears critical for the ability of UL144 to activate the CCL22 promoter in the presence of IE86.

FIG. 4.
Inhibition of UL144-mediated activation of the CCL22 promoter by IE86 is dependent upon the absence of a CREB binding site. HFF cells were transfected with pGL3-MDC (A and B) or deletion mutant −553 (lanes 5 to 8), −479 (lanes 9 to 12), ...

As our data suggest that inhibition of NF-κB by IE86 is due to the inhibition of the binding of NF-κB, we analyzed the occupancy of the NF-κB and CREB binding sites on the CCL22 promoter in the presence and absence of IE86 by DNA immunoprecipitation assays. Figure Figure4B4B shows that in the presence of IE86 alone, CREB is bound strongly to the CCL22 promoter, and there is a low but reproducible recruitment of NF-κB (Fig. (Fig.4B,4B, lanes 5 and 6), also consistent with a minor activation of the CCL22 promoter observed in the presence of IE86 (Fig. (Fig.3A3A and and4A).4A). In the presence of the UL144 effector alone, NF-κB is induced and becomes strongly associated with the promoter (Fig. (Fig.4B,4B, lanes 8 and 9), as expected. In the presence of both UL144 and IE86, there is no reduction in NF-κB binding to the promoter and both NF-κB and CREB bind effectively (Fig. (Fig.4B,4B, lanes 11 and 12). Figure Figure4C4C shows that consistent results were obtained with the endogenous CCL22 promoter. The assay is specific for promoters containing NF-κB and CREB binding sites, as PACT, an endogenous promoter that does not contain CREB or NF-κB binding sites, was not precipitated with anti-CREB or anti-NF-κB (Fig. (Fig.4D,4D, lanes 1 to 3, 5 to 7, 9 to 11, and 13 to 15). However, the endogenous PACT promoter was precipitated with anti-SP1, a transcription factor known to be associated with this promoter (Fig. (Fig.4D,4D, lanes 4, 8, 12, and 16). A slight enhancement of SP1 binding to the promoter in the presence of IE86 is consistent with previous findings (33). Furthermore, when the deletion mutant −289, which lacks the CREB binding domain, was tested for the ability to recruit CREB and IE86 (Fig. (Fig.4E),4E), CREB did not associate with the promoter, as expected (Fig. (Fig.4E,4E, lanes 2, 5, 8, and 11), and NF-κB binding was induced by the presence of UL144, also as expected (Fig. (Fig.4E,4E, compare lanes 3 and 9). However, the presence of IE86 prevented NF-κB binding to this CREB-deleted promoter (Fig. (Fig.4E,4E, lane 12). Consequently, IE86 had no effect on the binding of NF-κB to the full-length CCL22 promoter, but removal of the CREB binding site enabled IE86 to prevent UL144-mediated NF-κB binding to this promoter and to stimulate transcriptional activation.

To ensure that the differences in the CCL22 promoter activities in the presence of UL144 and IE86 were truly due to the absence of a CREB binding site, further experiments were carried out on CCL22 promoters which had site mutations in the κ and CREB binding sites (Fig. (Fig.5).5). Figure Figure5A5A (lanes 1 to 4) shows that in the luciferase assay, UL144 activated the WT CCL22 promoter and that it was not inhibited by IE86. However, if the two NF-κB binding sites are mutated, UL144 is unable to activate the CCL22 promoter (Fig. (Fig.5A,5A, lanes 5 to 8). In agreement with these studies, mutation of the two NF-κB binding sites prevented NF-κB from binding to the promoter but not IE86-induced binding of CREB (Fig. (Fig.5B).5B). In contrast, mutation of the CREB binding sites prevented CREB binding to the promoter (Fig. (Fig.5C)5C) and caused the CCL22 promoter to become sensitive to the presence of IE86 (Fig. (Fig.5A,5A, lanes 9 to 12).

FIG. 5.
Inhibition of UL144-mediated activation of the CCL22 promoter by IE86 is dependent upon the absence of a CREB binding site. HFF cells were transfected with pGL3-CCL22 with deletion mutations in the NF-κB (A and B) or CREB (A and C) binding sites ...

Taken together, these data show that UL144-mediated activation of NF-κB in the presence of IE86 is dependent upon the presence of a CREB binding site which enables UL144 to escape IE86-mediated negative regulation of the CCL22 promoter.

DISCUSSION

HCMV infection causes the upregulation of a variety of NF-κB-dependent transcripts in a temporal manner, reflecting the differential expression of both activatory and inhibitory functions of viral proteins during the time course of virus infection. For example, very early on in cytomegalovirus infection, interferon, a cytokine critically dependent on upregulation of both IRF3 and NF-κB, is induced (24). Subsequently, like many other viruses, HCMV then expresses proteins to inhibit this activation of interferon (20, 27). Our results show that IE86 blocks activation of the interferon promoter and some other NF-κB-responsive promoters, which is consistent with the already described inhibition of IFN expression at about 6 h postinfection (31).

However, at other times of infection it is clear that activation of NF-κB also occurs (10, 11, 25). This is, at least in part, due to expression of viral UL144, which is known to activate NF-κB, driving expression of the CCL22 chemokine (25). The results described here, together with our previous observations (25), show that infection with viruses carrying UL144 cause relocalization of the p65 subunit of NF-κB at 48 h postinfection. In contrast, viruses which lack UL144 due to a deletion of UL/b′ are unable to cause this nuclear relocalization of p65. Paradoxically, however, at the time of infection when CCL22 is being activated in an NF-κB-dependent manner, IE86 protein is still abundant in infected cells.

Clearly, IE86 is able to prevent UL144-mediated upregulation of a promoter which responds only to NF-κB (PRDII). In contrast, the UL144-mediated upregulation of CCL22, also mediated by NF-κB (25), is unaffected by the coexpression of IE86. However, the ability of IE86 to prevent UL144-mediated activation of the CCL22 promoter via NF-κB induction was conferred on the promoter by the removal of its CREB binding site.

IE86 does not prevent nuclear relocalization of the p65 subunit of NF-κB (31) (Fig. (Fig.1).1). Consequently, the mechanism by which IE86 prevents NF-κB-mediated activation of PRDII by UL144 is likely to involve the nuclear function of NF-κB, probably at the level of promoter binding, as has been shown for the IFN promoter (31).

Analysis of the UL144-mediated binding of NF-κB to the CCL22 promoter in the presence or absence of IE86 was entirely consistent with our promoter expression analyses. NF-κB binding to the CCL22 promoter was clearly induced by UL144 and was not affected by the coexpression of IE86. Intriguingly, this lack of effect of IE86 on NF-κB binding was drastically reversed when the CREB binding site within the CCL22 promoter was removed.

Clearly, the ability of IE86 to prevent NF-κB-mediated activation of NF-κB-responsive promoters is dependent on the promoter type, and in particular, on the context of NF-κB sites in the promoter with respect to other transcription factor binding sites. The presence or absence of specific transcription factor binding sites in any promoter plays a major role in tissue-specific and temporal regulation of promoter activity, but it is also becoming increasingly clear that the spacing and context of these sites may also be involved in suppressing or activating gene transcription (13, 32, 14, 17, 29).

Taken together, our data suggest a model whereby viral IE86 can indeed prevent NF-κB-mediated transcriptional activation, but only of certain types of NF-κB-responsive promoters. For other cellular promoters, such as the CCL22 promoter, the presence or absence of other transcription factor binding sites appears to play a profound role in preventing IE86-mediated repression of NF-κB-mediated activation.

We believe that these data may help explain conflicting observations on the activation of NF-κB-responsive gene expression during HCMV infection and suggest a mechanism for the differential ability of IE86 to regulate some, but not all, NF-κB-dependent cellular gene expression during the course of HCMV infection.

Acknowledgments

We thank Christopher Benedict (La Jolla Institute, La Jolla, CA) for the anti-UL144 rat monoclonal antibody, Matthew Reeves for reagents and assistance with the DNA immunoprecipitation protocols, and Yin Pang and Joan Baillie for technical support.

This work was funded by Wellcome Trust grant RG35737.

Footnotes

[down-pointing small open triangle]Published ahead of print on 20 February 2008.

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