Bcl-3-Regulated Transcription from Major Immediate-Early Promoter of Human Cytomegalovirus in Monocyte-Derived Macrophages

Reagents

Anti-p50, anti-p65, anti-RelB, anti-c-Rel, anti-p52, anti-Bcl-3, and the single-stranded NF-κB oligonucleotide and mutated oligonucleotide were purchased from Santa Cruz Biotechnology. Anti-IκBα, anti-phospho-IκBα, anti-IKKα, anti-IKKβ, anti-IKKγ, and anti-p- IKKγ were purchased from Cell Signaling Technologies; anti-pIKKα was purchased from Abcam, anti-p-IKKβ was purchased from US Biological, and peroxidase-conjugated secondary anti-rabbit and anti-mouse Ig were obtained from Jackson ImmunoResearch Laboratories. A scrambled control, p52, and Bcl-3 small interfering RNA (siRNA) duplex were purchased from Santa Cruz Biotechnology. Plasmid constructs p52 (pUNO-hNFkBp52a), p50 (pUNO-hNFkBp50), and p65 (pUNO-hNFkBp65) were purchased from Invivogen. Bcl-3-expressing plasmid (pCMV6-XL4-Bcl-3) and pCMV-Luc were obtained from Origene and PlasmidFactory, respectively. All other reagents were obtained from Sigma-Aldrich unless stated otherwise. Peripheral blood and human AB serum of a healthy donor was provided by Établissement Français du Sang (Bourgogne Franche-Comté, France). MRC5 human fibroblasts were obtained from BioMérieux. Cell culture flasks and cell scrapers were purchased from Nunc.

Isolation and culture of MDMs

PBMCs were isolated by Ficoll gradient centrifugation, as previously reported (35). Blood from a healthy donor was diluted with equal amounts of PBS, overlaid on Ficoll medium (Eurobio) and centrifuged at 900 × g for 30 min at 25°C. The PBMC band was removed and washed twice with PBS. Cell count was determined by Malassez cytometer (Poly Labo), and the cells were resuspended in serum-free RPMI 1640. The cells were plated in plastic cell culture flasks and incubated at 37°C. After 2 h, nonadherent cells were removed to enrich the culture for monocytes. Adherent cells (>95% CD14⁺ by flow cytometric analysis) were washed with sterile PBS and cultured in RPMI 1640 supplemented with 10% (v/v) human AB serum, penicillin (100 IU/ml), and streptomycin (100 μg/ml) for 7–10 days to allow the monocytes to differentiate into macrophages.

HCMV infection of MDMs and MRC5 fibroblasts

Cell-free virus stock was prepared by propagating four strains of HCMV (high-passage laboratory strain AD169 (AD169); a clinical isolate HCMV-DB; TB40/E; and TB40/F) in MRC5 human fibroblasts as previously described (36). AD169 is a highly passaged laboratory strain of HCMV originally isolated from the adenoids of a child (37). The clinical isolate HCMV-DB used in this study was isolated from a cervical swab specimen from a 30-year-old pregnant woman. Strains TB40/E and TB40/F have been developed by 22 passages in endothelial cells and fibroblasts, respectively (38). MRC5 human fibroblasts were cultured in MEM with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 μg/ml). Cells were infected with a viral isolate when the monolayer was confluent, and virus was collected when cytopathic effects were >90%. Supernatants were clarified by centrifugation and stored at −80°C until use. Virus titers were determined by plaque-forming assay in MRC5 human fibroblasts as previously described (39). The MDM cultures were infected at different multiplicities of infection (MOI) at days 7–10 postisolation, incubated for 12 h at 37°C, washed thoroughly, and covered with fresh medium.

Real-time PCR for quantification of viral titer

Quantification of viral titer in cell culture supernatants was performed by real-time PCR as previously described (36). DNA was extracted from 100 μl of supernatant using the KingFisher automatic instrument (Thermo Labsystems) and a QIAamp kit (Qiagen) according to the recommendations of the manufacturer. After elution in 100 μl of buffer, 5 μl of the DNA were used for PCR. HCMV DNA in the samples was quantified by real-time PCR using TaqMan technology on an ABI Prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer’s recommendations. The sequences of the primers used were 5′-AACATAAGGACTTTTCACACTTTT and 5′-GAATACAGACACTTAGAGCTCGGGT. The sequence of the TaqMan PCR probes was FAM 5′-CTGGCCAGCACGTATCCCAACAGCA-3′ TAMRA. Reaction samples had a final volume of 25 μl and contained 5 μl of extracted DNA and 20 μl of the mixture containing 12.5 μl of 2× TaqMan Universal PCR Master Mix (Applied Biosystems), 800 nM concentrations of each primer, and 200 nM TaqMan probe. An internal positive control, TaqMan Exogenous Internal Positive Control Reagents VIC Probe (Applied Biosystems), was included in each run to distinguish target negatives from PCR inhibition. A distilled water sample and a positive control with 3000 copies of HCMV DNA were processed in parallel with samples. Amplification conditions were 50°C for 2 min and 95°C for 10 min followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. To generate the HCMV external quantitative standard curve, PCR was performed on a plasmid containing one copy of the target sequence that had been serially diluted from 2 × 10⁶ to 2 × 10² copies/ml. Final quantification was performed with the comparative threshold value (C_T) method using the ABI Prim 7000 SDS software.

Isolation of nuclear and cytoplasmic extracts

Isolation of nuclear and cytoplasmic extracts was performed as previously described (40). Cells were scraped from the plastic surface of the culture dishes and washed with wash buffer (10 mM HEPES (pH 7.6), 10 mM KCl, 2 mM MgCl₂, 1 mM EDTA). Cell pellets were then incubated on ice with cytoplasmic isolation buffer (10 mM HEPES (pH 7.6), 10 mM KCl, 2 mM MgCl₂, 1 mM EDTA, 0.02% Nonidet P-40). Cytoplasmic extracts were collected by centrifugation, and the nuclear pellets were washed twice in wash buffer, spun, and incubated for 15 min on ice with nuclear isolation buffer (20 mM HEPES (pH 7.6), 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol). Supernatants containing nuclear extracts were collected by centrifugation and stored at −80°C. Protease inhibitors (1 mM DTT, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin) were added to all solutions. Protein concentration in nuclear and cytoplasmic extracts was determined by the Bradford method using a BioPhotometer (Eppendorf).

EMSA

To measure NF-κB activation, EMSA was conducted as previously described (41). Briefly, nuclear extracts prepared from HCMV-infected cells were incubated with 20 fmol of biotin-end-labeled 45-mer double-stranded NF-κB oligonucleotide, 5-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3 (bolded letters indicate NF-κB binding sites in the MIEP of HCMV) in the presence of binding buffer (10 mM Tris, 50 mM KCl, 1 mM DTT at pH 7.5 and polydeoxyinosinic-polydeoxycytidylic acid, 50 ng/μl). NF-κB oligonucleotide was labeled with biotin using the Biotin 3′ End DNA Labeling kit (Pierce), and complementary pairs were annealed by heating in boiling water for 5 min and then cooling slowly to room temperature. DNA-protein complexes were resolved from free oligonucleotide on a 6% native polyacrylamide gel in 1× Tris-borate-EDTA buffer using a MiniPROTEAN 3 Cell (Bio-Rad) and were transferred to a Biodyne precut nylon membrane (Pierce) using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Biotin-end-labeled DNA was detected using the LightShift Chemiluminescent EMSA kit (Pierce). A control Epstein-Barr nuclear Ag (EBNA) system (Pierce) containing biotin-EBNA control DNA and EBNA extract were assayed in parallel with the sample to ensure that the kit components and the overall procedure were working properly.

Microwell colorimetric NF-κB assay

Microwell colorimetric NF-κB assay was performed as described previously (42) using the Trans-AM NF-κB family transcription factor assay kit (Active motif). Briefly, cell extracts were incubated in a 96-well plate coated with an oligonucleotide containing the NF-κB consensus binding site (5′-GGGACTTTCC-3′). Activated transcription factors from extracts that bound specifically to the respective immobilized oligonucleotide were detected using Abs to NF-κB p50, p52, p65, RelB, and c-Rel subunits followed by a secondary Ab conjugated to HRP in an ELISA-like assay. Optical density was read within 5 min on a Multilabel Reader (PerkinElmer) at 450 nm. The specificity of the assay was validated by including both the wild-type and mutated oligonucleotides in the reaction. Raji nuclear extract was used a positive control.

Western blot

Cellular extracts of HCMV-infected cells were used to examine different protein expression by Western blot according to previously described procedures (40). Cellular extracts were resolved by 10% SDS-PAGE using a MiniPROTEAN 3 Cell (Bio-Rad). The proteins were electrotransferred onto a PVDF membrane (Amersham Biosciences) using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). The membranes were probed with primary Abs followed by HRP-conjugated secondary Ig raised against the appropriate species; bands were detected using the ECL Plus kit (Amersham).

Viral entry assay

Viral entry into MDMs and MRC5 fibroblasts was assayed as described previously (43). Cells were incubated at 37°C with HCMV-DB at MOIs of 1 and 10 for 2 h and washed three times with PBS. Cells were treated with 0.25% trypsin for 10 min to release the virions that had adhered to the surface but had not entered the cell. The cells were pelleted and washed once with serum neutralization solution and three times with PBS. DNA was extracted from the cell pellet using the KingFisher automatic instrument (Thermo Labsystems) and a QIAamp kit (Qiagen) according to the recommendations of the manufacturers. Samples of eluted DNA were analyzed by PCR using primers specific for the MIEP of HCMV (sense, 5′-TGGGACTTTCCTACTTGG-3′; antisense, 5′-CCAGGCGATCTGACGGTT-3′). The β-globin PCR gene was used as an internal control (sense, 5′-TCCCCTCCTACCCCTACTTTCTA-3′; antisense, 5′-TGCCTGGACTAATCTGCAAGAG-3′). The amplification products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.

Coimmunoprecipitation test

Total cellular extracts were prepared from uninfected and HCMV-infected primary macrophages 5 days postinfection. Cellular extracts were precleared with 50 μl of protein A-Sepharose 50% suspension (Amersham) for 1 h at 4°C. Bcl-3 and control Abs were added to the cleared supernatant and the mixture was incubated overnight at 4°C. Immune complexes were precipitated with 50 μl of protein A-Sepharose suspension and washed, and bound proteins were eluted with 30 μl of sample buffer. SDS-PAGE and Western blot were performed as described above using anti-p52 primary Ab.

Chromatin immunoprecipitation assay (ChIP)

ChIP assay was performed on HCMV-infected MDMs using EZ-Magna ChIP A (Upstate) according to a previously described procedure (44). Briefly, MDMs (0.5 × 10⁶ cells/reaction) were cross-linked with 1% formaldehyde and lysed, and nuclear extracts were sonicated to obtain DNA fragments ∼200–1000 bp long. After centrifugation, the nuclear extracts were diluted 10-fold with ChIP dilution buffer, and 1% of the material was saved as input. Nuclear extracts were incubated with 5 μg of control IgG, anti-Bcl-3, or anti-p52 Ab, and 20 μl of protein A magnetic beads slurry overnight at 4°C. Magnetic beads were separated and washed using a magnetic separator (Upstate). Immune complexes were eluted, and cross-linking was reversed by adding proteinase K and incubating at 62°C for 2 h. Immunoprecipitated DNA and input were analyzed by PCR using primers specific for the MIEP of HCMV as described above. The amplification product was run in a 2% agarose gel and visualized by ethidium bromide staining. Enrichment of MIEP was measured by real-time PCR using the ABI Prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer’s recommendations.

Transfection and luciferase assay

U937 cells (2 × 10⁶) were cotransfected with 20 μg of pCMV-Luc, which contains the luciferase reporter gene under control of MIEP, and a p52 plus Bcl3 or p50 plus p65 expression plasmid or the corresponding empty cloning vectors using the GenePulser XL electroporation system (Bio-Rad) according to manufacturer’s instructions. Luciferase activity in cell extracts was measured 24 h posttransfection using luciferase assay reagent (Promega) and a 20/20n Luminometer (Turner Biosystems). Luciferase activity was normalized to the total protein concentration and expressed as relative light units per microgram of protein. MRC5 fibroblasts were transfected with p52, Bcl-3, and p52 plus Bcl-3 expression plasmid using lipofectamine RNAiMAX (Invitrogen).

RNA interference

MDM cultures (0.5 × 10⁶ cells) were transfected with a scrambled control or p52 plus Bcl-3 or p50 plus p65 siRNA duplex (Santa Cruz Biotechnology) using Lipofectamine RNAiMAX (Invitrogen). MDMs were infected with the clinical isolate HCMV-DB 24 h posttransfection, and viral titers in culture supernatants were determined at the indicated times postinfection by real-time PCR, as described above. For monitoring knockdown, total cellular extracts were prepared daily for 3 days posttransfection. Expression of p52, Bcl-3, p50, and p65 protein was analyzed by Western blot as described above. Transfection efficiency was monitored using a fluorescein-conjugated scrambled control duplex and exceeded 50% in MDMs and 90% in MRC5 fibroblasts (data not shown).

Statistical analysis

Values are the means and SDs of independent experiments. Statistical analysis was performed by Student’s t test, and differences were considered significant at a value of p < 0.05. Microsoft Excel was used to construct the plots.

Low-level HCMV replication in primary MDMs vs efficient replication in fibroblasts

The ability of HCMV (AD169 and the clinical isolate HCMV-DB) to infect MDM and MRC5 fibroblast cultures was examined at different MOIs. MDM and MRC5 fibroblast cultures were infected as described in Materials and Methods, and supernatants were analyzed for production of extracellular virus by real-time PCR. The supernatants of the MDM culture infected with AD169 showed peak viral titer on day 8 postinfection at MOIs of 0.1 and 1. For the clinical isolate HCMV-DB, peak viral titer occurred at day 6 (MOI 0.1) and day 2 (MOI 1) postinfection (Fig. 1⇓, A–C). Supernatants of infected cultures assayed for 30 days showed similar growth patterns (data not shown). Moreover, no cytopathic effects were observed in the infected MDMs. In contrast, the viral titers in culture supernatants from MRC5 fibroblasts infected with the same viral isolates were much higher than those from MDM cultures (Fig. 1⇓, E–G). To assess the possibility that blocked viral entry influenced the differences in the viral titers, viral entry was assayed in both MDMs and MRC5 fibroblasts. As shown in Fig. 1⇓, D and H, viral entry was similar in both cell types. To rule out the possibility that cell death contributed to the low viral production by MDM cultures, infected MDMs were examined for viability at 5 and 10 days postinfection by trypan blue exclusion. There were no differences in trypan blue exclusion between the control and infected groups; <1% dead cells were observed in the control and infected groups (data not shown), indicating that the inability of the culture to support high viral replication was not due to MDM death. To assess the differential growth of HCMV in MDMs vs MRC5 fibroblasts, expression of HCMV pp65 protein was assayed 5 days postinfection in MDMs and MRC5 fibroblasts infected with HCMV-DB. HCMV pp65 protein was expressed in 1–2% of MDM cells, whereas most of the cells in the MRC5 fibroblast cultures expressed this protein (data not shown).

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FIGURE 1.

Growth curve of HCMV in MDMs vs MRC5 fibroblasts. The MDM and the MRC5 cultures were left uninfected or infected at indicated MOIs with laboratory strain AD169 (A and E) and the clinical isolate HCMV-DB (B and F). Viral titers in culture supernatants were determined at the indicated times postinfection by real-time PCR. Peak viral titers in supernatants after infection of MDMs (C) and MRC5 fibroblasts (G) with laboratory strain AD169 and the clinical isolate HCMV-DB. For each point, mean values ± SD of duplicate samples are shown. Noninfected MDMs (D) and MRC5 fibroblasts (H) and cultures infected at the indicated MOI for 2 h were treated with trypsin for 10 min and then washed. Samples of extracted DNA were analyzed by PCR using primers specific for the MIEP of HCMV and for β-globin (internal loading control). The amplification products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. IE, Immediate-early Ag.

Subsequently, the growth of the clinical isolate HCMV-DB in MDMs was compared with that of the endothelial cell-propagated HCMV strain (TB40/E) and the fibroblast-propagated strains (AD169 and TB40/F). The endothelial cell-propagated strain TB40/E has been reported to replicate efficiently in MDMs (45). The clinical isolate HCMV-DB and the TB40/E strain grew better in MDMs than did the fibroblast-propagated strains (Fig. 2⇓).

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FIGURE 2.

Growth of the clinical isolate HCMV-DB, endothelial cell-propagated HCMV strain (TB40/E), and fibroblast-propagated strains (TB40/F and AD169) in MDM cultures. The MDM cultures were left uninfected or infected at an MOI of 1 with different HCMV strains; viral titers in culture supernatants were determined at the indicated times postinfection by real-time PCR. For each point, mean values ± SD of duplicate samples are shown.

Activation of the noncanonical NF-κB pathway in HCMV-infected MDMs

Because HCMV infection of fibroblasts and monocytes results in activation of transcription factor NF-κB (22, 23) and because the NF-κB signaling pathway potentially contributes to the HCMV replication strategy (24, 25, 26, 27), we evaluated the activation and composition of NF-κB complexes in MDMs and fibroblasts infected with the clinical isolate HCMV-DB. At 5 days postinfection, EMSA demonstrated that infection of MDMs and MRC5 fibroblasts with HCMV resulted in activation of NF-κB (Fig. 3⇓, A and F). The gel shift bands were specific; formation of the complex was diminished by inclusion of an unlabeled NF-κB oligonucleotide but not by inclusion of a mutated NF-κB oligonucleotide (data not shown). To identify the subunits in the NF-κB complexes, nuclear extracts from HCMV-infected MDMs and MRC5 fibroblasts were incubated with Abs against different NF-κB subunits and against Bcl-3, and DNA-binding activity was assayed by EMSA. Interference of NF-κB activation by anti-p52 Ab and anti-Bcl-3 Ab (Fig. 3⇓B) suggested that p52 and Bcl-3 were the major components of the activated NF-κB complex in HCMV-infected MDMs. NF-κB subunit binding activity was also evaluated by microwell colorimetric NF-κB assay using Abs against NF-κB subunits (p50, p52, p65, RelB, and c-Rel). The results revealed an increase in the binding activity of p52 in HCMV-infected MDMs 5 days postinfection (Fig. 3⇓C). Overall, the results obtained by both techniques indicate that activated NF-κB complexes in HCMV-DB-infected MDMs involve the NF-κB subunit p52 (which is known to bind to NF-κB binding sites as a homodimer; see Ref. 7, 46) and Bcl-3 (which is known to bind with p52 and p50 homodimers; see Ref. 8), suggesting a p52/Bcl-3 complex. Recent studies have demonstrated the activation of the canonical p50/p65 NF-κB complex in monocytes early after HCMV binds to cell membrane receptors (47, 48). Therefore, we assayed the composition of NF-κB early after infection and found that the canonical p50/p65 NF-κB complex was activated during early phases of MDM infection by HCMV (Fig. 3⇓C), with a shift to the p52/Bcl-3 complex occurring later during infection.

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FIGURE 3.

Activation and composition of NF-κB complexes in MDMs and in MRC5 fibroblasts after HCMV infection. MDM cultures (A) and MRC5 fibroblasts (F) were infected with the clinical isolate HCMV-DB at an MOI of 1. Nuclear extracts were prepared at 5 days postinfection and then assayed for NF-κB activation by EMSA. B and G, Interference of NF-κB activation in response to HCMV-DB by Abs against NF-κB subunits: Nuclear extracts from infected MDMs (B) and MRC5 fibroblasts (G) were incubated for 20 min with anti-p50, anti-p52, anti-p65, anti-c-Rel, anti-RelB, and anti-Bcl-3 Abs and then assayed for NF-κB binding activity by EMSA. C and H, Nuclear extracts from HCMV-DB-infected MDMs (C) and MRC5 fibroblasts (H) were assayed at 6 h and 5 days postinfection for NF-κB binding subunits by Microwell colorimetric NF-κB assay. NF-κB activation is shown in fold increase relative to NF-κB binding activity in uninfected cells (set equal to 1). Mean values ± SD of two independent experiments are shown. D and I, IκBα phosphorylation and degradation in HCMV-DB-infected MDMs and MRC5 fibroblasts. Cytoplasmic extracts were prepared from infected MDMs (D) and MRC5 fibroblasts (I) at 5 days postinfection and then assayed for the expression of phospho-IκBα and IκBα by Western blot. β-Actin was used as a loading control. E and J, IKKα, IKKβ, and IKKγ expression and phosphorylation in HCMV-DB-infected MDMs and MRC5 fibroblasts. Cytoplasmic extracts were prepared from infected MDMs (E) and MRC5 fibroblasts (J) at 5 days postinfection and then assayed for the corresponding protein by Western blot. β-Actin was used as a loading control.

In contrast to the NF-κB complexes observed in MDMs, anti-p50, anti-p65, and the combination of both Abs diminished the binding activity of NF-κB in HCMV-infected MRC5 fibroblasts (Fig. 3⇑G), suggesting the presence of canonical p50/p65 heterodimers in the activated NF-κB complex in HCMV-infected fibroblasts. Thus, NF-κB subunit binding activity was evaluated by microwell colorimetric NF-κB assay. Nuclear extracts from infected fibroblasts showed increased binding activity of p50 and p65 (Fig. 3⇑H), confirming the results obtained by EMSA.

In addition, expression of IκBα and a phosphorylated form of IκBα, which is required for IκBα degradation, was monitored by Western blot. We observed decreased phosphorylation of IκBα in infected MDMs, whereas the expression of IκBα increased (Fig. 3⇑D). In contrast, HCMV infection of fibroblasts resulted in phosphorylation and degradation of IκBα, as typically occurs during activation of the canonical NF-κB pathway (Fig. 3⇑I). Moreover, expression and phosphorylation of upstream IKKα, IKKβ, and IKKγ were monitored by Western blot.

Although there were no differences in the expression levels of IKKα (Fig. 3⇑, E and J), IKKα was highly phosphorylated in HCMV-infected MDMs (Fig. 3⇑E) as compared with MRC5 fibroblasts (Fig. 3⇑J). In contrast, phosphorylation of IKKβ was observed only in infected MRC5 fibroblasts (Fig. 3⇑, E and J). Phosphorylation of IKKγ was not observed in either cell type.

Bcl-3 expression in HCMV-infected MDMs and activation of MIEP by the p52/Bcl-3 complex

Bcl-3 has the potential to activate transcription after association with p52 and p50 (8, 9, 11); therefore, we wanted to evaluate the presence of p52/Bcl-3 complexes after HCMV infection of MDMs. We assayed Bcl-3 expression by MDMs and MRC5 fibroblasts after HCMV-DB and AD169 infection by Western blot and found that the amount of Bcl-3 was increased in nuclear extracts of infected MDMs but not in those of MRC5 fibroblasts (Fig. 4⇓A). In addition, p65 expression was observed early after HCMV infection of MDMs but not later on (Fig. 4⇓A), indicating a shift from p65 to Bcl-3 in MDMs. On the other hand, p65 was expressed both early and late during HCMV infection of MRC5 fibroblasts (Fig. 4⇓A). Quantification of Bcl-3 and p65 protein expression by densitometry showed that Bcl-3 expression and the Bcl-3/p65 ratio was much higher in infected MDMs than in infected MRC5 fibroblasts (Fig. 4⇓B). Next, we analyzed the interaction between Bcl-3 and p52 by coimmunoprecipitation assay. Total cellular extracts from uninfected and HCMV-DB-infected MDMs were immunoprecipitated with control IgG and anti-Bcl-3 Ab, followed by detection with anti-p52 Ab. The results revealed the specific interaction of Bcl-3 with p52 in HCMV-infected MDMs (Fig. 4⇓C).

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FIGURE 4.

Bcl-3 up-regulation and binding with p52 in HCMV-infected MDMs. A, MDM and MRC5 fibroblast cultures were left uninfected or infected with the clinical isolate HCMV-DB and the laboratory strain AD169 at an MOI of 1. Nuclear extracts were prepared at different days postinfection and then assayed for the expression of Bcl-3 and p65 by Western blot. β-Actin was used as a loading control. B, Activation of Bcl-3 and p65 was quantified using ImageJ 1.40 software (National Institutes of Health) in uninfected and HCMV-DB-infected MDMs. Results are shown as relative intensities of Bcl-3 and the Bcl-3:p65 ratio. Means ± SD of two independent experiments are shown. C, Bcl-3 interacts with p52 in HCMV-infected MDM. Total cellular extracts from uninfected and HCMV-DB-infected MDMs were immunoprecipitated with anti-Bcl-3 and control Abs. Immunoprecipitated material was analyzed by Western blot using an anti-p52 Ab. The input control represents 1% of the material used for immunoprecipitation.

Next, we examined the ability of p52 and Bcl-3 to regulate transcription from HCMV MIEP by luciferase reporter assay using pCMV-Luc, in which the luciferase reporter gene is under the control of the MIEP of HCMV (Fig. 5⇓A). Promonocytic U937 cells were transfected with p52 plus Bcl3 or p50 plus p65-expressing plasmids along with pCMV-Luc; luciferase activity in cell extracts was measured 24 h posttransfection. We found that p52/Bcl-3 could activate transcription from MIEP (Fig. 5⇓B) but that the ability of p65 or the p50/p65 complex to drive transcription of the HCMV MIEP-luciferase reporter construct was many fold greater than that of p52/Bcl-3 (Fig. 5⇓C), which is in line with the low-level growth of the virus in MDMs vs efficient growth in fibroblasts. Next, we analyzed whether the MIEP was occupied by p52 and Bcl-3. The presence of p52 and Bcl-3 on the MIEP of HCMV-DB in acutely infected MDMs was demonstrated by ChIP using Abs specific for p52 and Bcl-3 (Fig. 6⇓, A and B). In addition, we assayed the binding of p50 and p65 with MIEP on days 1 and 5 postinfection. The results demonstrated that the canonical p50/p65 complex was activated early but not late after infection (Fig. 6⇓C). Finally, MDM and MRC5 fibroblast cultures were transfected with p52 plus Bcl-3 and p50 plus p65-specific siRNA and infected with HCMV-DB. Knockdown of p52, Bcl-3, p50, and p65 proteins in MDMs (Fig. 7⇓A) and fibroblasts (Fig. 7⇓B) was monitored by Western blot. Band density was quantified using ImageJ 1.40 software (National Institutes of Health), and the results are shown as relative intensities (Fig. 7⇓C). Knockdown of p50/p65 resulted in decreased growth of HCMV-DB in MDM cultures on day 1, whereas p52/Bcl-3 ablation resulted in decreased viral growth on days 4–6 postinfection (Fig. 8⇓A). In addition, p52/Bcl-3 knockdown did not block viral replication in MRC5 fibroblasts whereas p50/p65 ablation resulted in decreased growth of HCMV-DB in fibroblasts (Fig. 8⇓B). Cultures infected with AD169 showed similar results (data not shown). Moreover, overexpression of p52 and Bcl-3 in HCMV-infected MRC5 fibroblasts resulted in reduced viral replication (Fig. 8⇓C). Altogether, our results indicate that in HCMV-infected MDMs, HCMV MIEP is preferentially activated by a p52/Bcl-3 complex.

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FIGURE 5.

Activation of gene expression from MIEP by NF-κB subunits. A, Schematic presentation of the luciferase reporter plasmid. Luciferase expression is under control of MIEP, which contains four NF-κB binding sites. B–C, U937 cells (2 × 10⁶) were transfected with p52 plus Bcl3 (B) and p50 plus p65 (C) expression plasmids along with pCMV-Luc, which contains the luciferase gene under the control of MIEP of HCMV. Luciferase activity was measured in cell extracts at 24 h posttransfection. Data are expressed as relative light units (RLU) per microgram of protein. Controls were transfected with the corresponding empty vectors. Mean values ± SD of two independent experiments are shown; ∗, p < 0.05.

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FIGURE 6.

Bcl-3 and p52 binding to MIEP in HCMV-infected MDMs. A, ChIP assay was performed with HCMV-DB-infected MDM cultures at 5 days postinfection. Briefly, cross-linked cell extracts from MDMs infected with HCMV-DB at an MOI of 1 were immunoprecipitated with control IgG, Bcl-3, and p52 Abs and assayed for the presence of MIEP by PCR. Amplification products were resolved by 2% agarose gel electrophoresis. Normal rabbit IgG was used as a nonspecific binding control. Input represents 1% of material used for immunoprecipitation. B, Enrichment of MIEP with p52 and Bcl-3 was measured by real-time PCR. Results represent mean values ± SD of two independent experiments. C, Enrichment of MIEP by p50 and p65 was measured by real-time PCR at day 1 and day 5 postinfection. Results represent mean values ± SD of two independent experiments.

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FIGURE 7.

Knockdown of p52, Bcl-3, p50, and p65 proteins by siRNA. MDM (A) and MRC5 fibroblast (B) cultures were transfected with scrambled control, p52, Bcl-3, p50, or p65 siRNA, and total cellular extracts were prepared up to 3 days posttransfection. Protein expression was analyzed by Western blot; β-actin was used as a loading control. C, Protein levels after siRNA transfection were quantified by densitometry using ImageJ 1.40 software (National Institutes of Health), and the results are presented as relative intensities. Means ± SD of two independent experiments are shown.

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FIGURE 8.

Effect of p52 plus Bcl-3 and p50 plus p65 siRNA on HCMV replication. MDM (A) and MRC5 fibroblast (B) cultures (0.5 × 10⁶ cells) were transfected with a scrambled control, p52 plus Bcl-3 (top), or p50 plus p65 (bottom) siRNAs and infected with HCMV-DB at an MOI of 0.1. C, Inhibition of HCMV replication in p52 plus Bcl-3-transfected fibroblasts. p52, Bcl-3, and p52 plus Bcl-3 were overexpressed in MRC5 fibroblasts by transfection with the corresponding expression plasmid and infected with HCMV-DB at an MOI of 0.1. Viral titers in culture supernatants were determined at the indicated times postinfection by real-time PCR. An uninfected culture was tested in parallel. For each point, means ± SD of duplicate samples are shown; ∗, p < 0.05.