The Human Cytomegalovirus U_L26 Protein Antagonizes NF-κB Activation

Cell culture, viruses, and chemicals.

MRC5 fibroblasts (passages 23 to 29) were cultured in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum. The wild-type (WT) HCMV strain used in this study was BADwt, a bacterial artificial chromosome (BAC) clone of Ad169 (26, 27). The ΔU_L26 mutant in these studies is a BADwt derivative that was previously described (15, 28). Viral stocks were prepared through combining clarified infected cell medium (3,000 × g) with the remaining clarified infected-cell lysate. For the lysate, the scraped cells were resuspended in 5 ml of infected media, sonicated, and clarified through centrifugation at 3,000 × g. Titers of all stocks were determined by plaque assay. For all infections, cells were grown to a confluence of ∼3.2 × 10⁴ cells per cm² prior to infection. Once confluent, medium was removed and serum-free medium was added for 24 h. In all infections, viral inocula were added to cells for a 2-h adsorption period and then the viral inocula were aspirated. For all experiments at a multiplicity of infection (MOI) of 3.0, the extent of infection was monitored through analysis of either green fluorescent protein (GFP) fluorescence (for the ΔU_L26 mutant that expresses GFP in place of U_L26 [15]) or through the appearance of HCMV's distinctive cytopathic effect. In either case, based on these measures, infection at an MOI of 3.0 resulted in ∼100% infection rates. In experiments utilizing UV-irradiated virus, the viral inocula were exposed to 254-nm light at 0 or 1,920 mJ/cm² with a model 2400 Stratalinker UV cross-linker prior to infection. TNF-α was purchased from Sigma, and alpha interferon (IFN-α) was purchased from PBL Assay Science.

Plaque formation assay.

Serum-starved confluent MRC5 fibroblasts were treated with TNF-α or IFN-α at various concentrations in 12-well dishes. The cells were then incubated at 37°C for 4 h prior to infection. After removal of the cytokine-containing medium, a fixed number of PFU from freshly thawed virus stocks was seeded. The infection was followed by a conventional plaque assay gel overlay. At 10 days postinfection (dpi), the number of plaques in each well was counted. The number of plaques at each cytokine concentration was plotted as a percentage of the non-cytokine-treated control. Linear or exponential regression analysis was subsequently performed using Origin 8. The resulting best-fit curves were utilized to extrapolate 50% inhibitory concentrations (IC₅₀).

Confocal microscopy.

For analysis of endogenous RelA, RelB, and IRF3 localization, serum-starved MRC5 fibroblasts were grown on glass coverslips. For RelA analysis, TNF-α was added at 48 h postinfection (hpi) for 1 h. At various time points postinfection, cells were washed once with phosphate-buffered saline (PBS), fixed with 2% paraformaldehyde in PBS for 20 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 and 0.1% SDS for 15 min, and washed twice with PBS containing 0.05% Tween 20. For IRF3, at 6 hpi, cells were fixed and permeabilized. Cells were subsequently blocked by overnight incubation in PBS containing 2% bovine serum albumin (BSA), 5% goat serum, 5% human serum, and 0.3% Triton X-100. Cells were incubated with primary antibody to RelA (C-20; Santa Cruz) or RelB (C-19; Santa Cruz), diluted in PBS plus 0.05% Tween 20 for 1 h, washed with PBS containing 0.01% Tween 20 three times, incubated with fluorochrome-conjugated anti-rabbit secondary (Invitrogen) antibody for 1 h, and washed three times in the same buffer lacking antibody. Coverslips were mounted in SlowFade Gold antifade reagent (Molecular Probes) and DAPI (4′,6′-diamidino-2-phenylindole). Confocal images were captured with an FV1000 Olympus laser scanning confocal microscope. All images were captured under identical confocal settings.

For confocal analysis of endogenous RelA with U_L26 transfection, 293T cells were transfected with either 2 μg pAC empty vector or 2 μg pAC-UL26 expression plasmid using Oligofectamine (Invitrogen). At 24 h posttransfection, cells were treated with 10 ng/ml TNF-α for 24 h. Cells were washed once with PBS, fixed, permeabilized, and blocked as indicated above prior to immunostaining and confocal analysis. For confocal analysis of RelA and IRF3, 30 to 40% confluent 293T cells were cotransfected with 2 μg of either pCAGGS GFP-RelA or pEGFP-C1-hIRF3 and 2 μg of pAC empty vector or pAC-UL26 expression plasmid. At 24 hpi, cells were treated with 10 ng/ml TNF-α for 24 h. Cells were then washed once with PBS, fixed, permeabilized, and blocked as indicated above prior to immunostaining and confocal analysis. The IRF3 (17C2) antibody was purchased from KeraFAST.

For confocal analysis of RelA and IRF3 at various times post-TNF-α treatment, 30 to 40% confluent 293T cells were cotransfected with 2 μg of either pCAGGS GFP-p65 or pEGFP-C1-hIRF3 and 2 μg of pAC empty vector or pAC UL26 overexpression plasmid. At 24 hpi, cells were treated with 10 ng/ml TNF-α for various time periods. GFP-positive cells were counted using a Nikon Eclipse TE200 microscope. The percentage of cells containing nuclear GFP was calculated in comparison to the total number of cells that contained detectable GFP.

NF-κB and IRF3 luciferase assays.

TNF-α or Sendai virus (SeV) induction of the NF-κB-dependent reporter plasmid pNF-κB–FF (RelA-specific vector) was done as described previously (29). SeV induction of the IRF3-dependent reporter plasmid (p55C1B-FF) was described previously (30). Briefly, 30 to 40% confluent 293T cells in 6-well plates were cotransfected using calcium phosphate (Stratagene) with 1 μg of pNFκB-FF or 1 μg p55C1B-FF, 1 μg of pAC empty vector, or 1 μg pAC U_L26 overexpression vector (0.25, 0.5, or 1.0 μg for the dose-dependent assay) and 1 μg of an expression plasmid encoding Renilla luciferase (RL) under the control of a simian virus 40 promoter (pSV40-RL) to normalize transfection efficiencies. For TNF-α-mediated NF-κB activation experiments, cells were treated 24 h posttransfection with 10 ng/ml TNF-α for 24 h, before harvesting for analysis of luciferase activity. For SeV infections, cells were mock or SeV infected (MOI = 3.0) for 1 h at room temperature in 1× PBS, and cell lysates were prepared 16 to 18 h posttreatment. Luciferase activities were determined using a Promega dual-luciferase reporter assay and a Lumicount luminometer (Hewlett Packard). Reporter gene activation was calculated relative to RL activity to control for transfection efficiency.

Real-time qPCR.

At various times postinfection, medium was aspirated from cells, total RNA was extracted with TRIzol, and cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen). Quantitative PCR (qPCR) was performed using Fast SYBR green master mix, a model 7500 Fast real-time PCR system, and Fast 7500 software (Applied Biosystems) according to the manufacturer's instructions. Gene expression levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined according to the 2^−ΔΔCT method. Specific primer pairs used are as follows: interleukin-6 (IL-6), 5′-AAA-TTC-GGT-ACA-TCC-TCG-ACG-GCA-3′ (forward) and 5′-AGT-GCC-TCT-TTG-CTG-CTT-TCA-CAC-3′ (reverse); IL-8, 5′-AGA-AAC-CAC-CGG-AAG-GAA-CCA-TCT-3′ (forward) and 5′-AGA-GCT-GCA-GAA-ATC-AGG-AAG-GCT-3′(reverse); GAPDH, 5′-CAT-GTT-CGT-CAT-GGG-TGT-GAA-CCA-3′ (forward) and 5′-ATG-GCA-TGG-ACT-GTG-GTC-ATG-AGT-3′ (reverse).

Protein gel electrophoresis and Western blot analysis.

Protein from cell lysates was solubilized in disruption buffer (50 mM Tris [pH 7.0], 2% SDS, 5% 2-mercapoethanol, and 2.75% sucrose), separated by either 10% or 15% SDS-PAGE, and transferred to nitrocellulose in Tris-glycine transfer buffer. Blots were then stained with Ponceau S to visualize protein bands and ensure equal protein loading. The membranes were blocked in 5% milk in Tris-buffered saline-Tween 20 (TBST), followed by incubation in primary antibody. After subsequent washes, blots were treated with secondary antibody and protein bands were visualized using the enhanced chemiluminescence (ECL) system (Pierce). The primary antibodies were specific for IκBα (Cell Signaling), cellular protein tubulin (Epitomics), phosphor-IKKα(Ser176)/IKKβ(Ser177) (Cell Signaling), total IKKα/β (Santa Cruz), NF-κB p50 (H-119; Santa Cruz), p52/P100 (18D10; Cell Signaling), GAPDH (Cell Signaling), and the viral protein U_L26 (7H19) (31). The secondary antibodies were rabbit polyclonal (Santa Cruz Biotechnology, Inc.) and mouse monoclonal (Abcam). Quantification of the relative abundance of Western blot bands was performed using a ChemiDoc XRS+, a charge-coupled-device (CCD)-based fluorescent quantification system, and ImageLab software, both from Bio-Rad. All Western blot bands were within the linear range of the CCD camera.

ELISA.

The quantities of IL-6 and TNF production in cells were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits (PeproTech) by following the manufacturer's instructions. Values are means plus standard errors (SE; n = 3 replicates).

Statistical analysis.

For all of the Western analyses, a representative blot from at least 3 independent experiments is shown, with the exception of those shown in Fig. 3C and D, which are representative of 2 independent experiments. For all of the Western quantifications, the data are expressed as averages plus standard errors of the means (SEM) from at least two independent biological replicates, which were subsequently analyzed technically in duplicate, i.e., a separate SDS-PAGE gel, blot, and ChemiDoc scan. For all luciferase experiments, the data are representative of at least three biological replicates. Where indicated, the significance of the results was assessed by Student's t test. A P value of <0.05 was considered statistically significant.

A U_L26-deletion mutant activates the noncanonical NF-κB pathway.

HCMV appears to have a complex relationship with the NF-κB pathway. Several reports indicate that HCMV can induce NF-κB activity (32 – 37), while others indicate that HCMV antagonizes NF-κB activation (12, 38, 39). To explore whether U_L26 plays a role in HCMV's modulation of NF-κB activity, we examined the localization of the RelA NF-κB subunit after infection with WT HCMV or the ΔU_L26 mutant. Consistent with previous reports (35), WT HCMV induced higher levels of RelA than those seen with mock-infected cells (Fig. 1A and B). A similar induction of RelA was observed with ΔU_L26-infected cells (Fig. 1A and B). In canonical NF-κB signaling, RelA dimerizes with NF-κB p50, which is a proteolytic product of a p105 protein precursor (22). The total amounts of p105 and its processed p50 product were similar between WT and ΔU_L26 infection, although both were enhanced in comparison to results with mock-infected cells (Fig. 1B). Further, there was little difference between WT- and ΔU_L26-infected cells with respect to RelA or p50 localization. RelA localized primarily to the cytoplasm (Fig. 1A), where it is transcriptionally inactive, while p50 localized primarily in the nucleus in both mock- and HCMV-infected cells, regardless of whether U_L26 was present (Fig. 1C). Thus, our results indicate that deletion of U_L26 had little impact on the levels and localization of the canonical NF-κB subunits RelA and p50.

In contrast to RelA, immunofluorescence of RelB differed substantially between WT and ΔU_L26 infection (Fig. 1D). At 4 h post-ΔU_L26 infection, RelB was primarily localized in the nucleus, in contrast to results with WT infection. A similar trend was observed at 48 hpi, where RelB immunofluorescence was almost entirely nuclear during ΔU_L26 infection in contrast to results with WT infection (Fig. 1D). Infection with both WT and ΔU_L26 viruses induced higher RelB levels than those for mock-infected cells, with ΔU_L26-infected cells exhibiting an increase in RelB in comparison to results for WT-infected cells (Fig. 1E). This increase was most notable at 48 hpi, a time at which RelB levels in ΔU_L26-infected cells were >2-fold higher than those in WT-infected cells (Fig. 1E). The NF-κB p100 protein is processed to generate the p52 subunit which forms the noncanonical NF-κB dimer with RelB (22). Both WT and ΔU_L26 infections induced higher levels of p100 than those for mock-infected cells (Fig. 1E), but the deletion of U_L26 had little impact on p52 levels or localization relative to results with the WT (Fig. 1E and F, respectively). The finding that ΔU_L26 infection induces RelB levels and nuclear translocation suggests that deletion of U_L26 induces noncanonical NF-κB activation.

To examine the potential impact of RelB nuclear translocation during ΔU_L26 infection, we analyzed the mRNA levels of IL-6 and IL-8, genes known to be regulated by NF-κB (40 – 43). Infection with the ΔU_L26 mutant substantially induced accumulation of IL-6 mRNA in comparison to either mock or WT infection (Fig. 2A), which is consistent with increased NF-κB activation during ΔU_L26 infection. In contrast, the levels of IL-8 mRNA at 24 hpi were actually reduced during ΔU_L26 infection, but they were not significantly different between WT and ΔU_L26 infection at 48 or 72 hpi (Fig. 2B). To determine whether the induction of IL-6 mRNA observed during ΔU_L26 infection correlates to an induction of IL-6 production, we measured the amount of IL-6 produced during viral infection. Consistent with the mRNA data, infection with the ΔU_L26 mutant significantly increased cellular IL-6 secretion over that seen with WT-infected cells (Fig. 2C). Combined, these results indicate that the deletion of U_L26 induces RelB nuclear translocation, and the induction of IL-6 expression and secretion.

A U_L26-deletion mutant is more sensitive than the WT to treatment with antiviral cytokines.

Since the ΔU_L26 mutant induces RelB nuclear translocation indicating noncanonical NF-κB activation, we hypothesized that the ΔU_L26 mutant might be sensitive to innate immune challenge. To explore whether U_L26 is important for modulation of innate immunity, we compared the abilities of WT HCMV and a U_L26-deletion mutant (ΔU_L26) to initiate infection after challenge with NF-κB activating cytokines. After cells were treated with various concentrations of TNF-α or IFN-α, they were infected with an equivalent number of WT or ΔU_L26 PFU. Increasing concentrations of antiviral cytokines reduced the ability of either virus to initiate a productive infection as assessed by plaque formation, with TNF-α treatment inhibiting the establishment of infection at substantially lower concentrations than those for IFN-α (Fig. 2D and E). For both cytokines tested, the ΔU_L26 virus was more sensitive to treatment than WT HCMV (Fig. 2D and E). However, the sensitivity of ΔU_L26 was more pronounced in response to TNF-α than IFN-α. After TNF-α treatment, the ΔU_L26 mutant exhibited a >2.5-fold decrease in the IC₅₀ of plaque formation (P < 0.01) (Fig. 2D and E). To explore the possibility that deletion of U_L26 might impact TNF-α production, we assayed for TNF-α levels after mock, WT, or ΔU_L26 infection. WT HCMV infection induced more TNF-a production than mock-infected cells (Fig. 2F), which agrees with previous reports (44, 45). Cells infected with ΔU_L26 induced a similar amount of TNF-α (Fig. 2F), suggesting that increased TNF-α production does not explain the increased sensitivity of ΔU_L26 virus. Rather, these results suggest that in cells exposed to TNF-α, the U_L26 protein is important for initiation of infection.

The HCMV U_L26 protein antagonizes TNF-α-induced NF-κB activation.

One of the major consequences of TNF-α treatment is NF-κB pathway activation (reviewed in reference 46). Given that the ΔU_L26 mutant induces noncanonical NF-κB activity as well as sensitivity to TNF-α treatment, we wanted to explore potential mechanistic links between these phenotypes. We tested the impact of U_L26 deletion on NF-κB activity after challenge with TNF-α. After TNF-α challenge, RelA localized to the nucleus in mock-infected cells (Fig. 3A). Infection with WT HCMV blocked TNF-α-induced RelA nuclear translocation (Fig. 3A). In contrast, ΔU_L26-infected cells failed to block TNF-α-induced RelA nuclear accumulation (Fig. 3A). While WT HCMV infection blocked RelA nuclear translocation upon TNF-α treatment (Fig. 3A), it failed to block RelB translocation upon TNF-α treatment (Fig. 3B). Interestingly, TNF-α treatment induced more RelB nuclear translocation in WT-infected cells than in mock-infected cells (Fig. 3B). As expected, ΔU_L26 infection induced RelB nuclear translocation regardless of whether cells had been treated with TNF-α.

As RelA nuclear translocation is induced by IκB degradation, we examined IκB levels during HCMV infection. In the absence of TNF-α treatment, infection with WT HCMV or ΔU_L26 induced an increase in total IκBα levels relative to results seen with mock infection (Fig. 3C). However, there was little difference in total IκBα levels in WT- or ΔU_L26-infected cells, with the exception of 72 hpi, when there was more IκBα present in WT- than in ΔU_L26-infected cells (Fig. 3C). With TNF-α treatment, there was little difference in the amount of IκBα levels between WT- and ΔU_L26-infected cells at 24 hpi (Fig. 3D). In contrast, at 48 hpi, TNF-α treatment induced significantly more IκBα degradation in ΔU_L26-infected and mock-infected cells than in cells infected with WT HCMV (Fig. 3D). These results suggest that the U_L26 protein is important for HCMV-mediated inhibition of TNF-α-induced NF-κB activity inasmuch as its deletion results in increased RelA nuclear translocation and IκB degradation.

While we found the U_L26 protein to be necessary for HCMV-mediated inhibition of TNF-α-induced RelA nuclear translocation, we wanted to test whether it was sufficient to block TNF-α-induced NF-κB activity in the absence of other HCMV viral proteins. Expression of U_L26 blocked TNF-α-induced NF-κB-dependent luciferase activity (Fig. 4A). The inhibition of TNF-α-induced NF-κB activity was largely U_L26 dose dependent (Fig. 4B). To determine whether U_L26 expression was sufficient to block NF-κB nuclear translocation upon TNF-α treatment, we examined the localization of GFP-tagged RelA. Shortly after TNF-α treatment, GFP-RelA migrated to the nucleus in vector control-transfected cells (Fig. 4C). TNF-α-induced GFP-RelA translocation was largely blocked by coexpression of the U_L26 protein (Fig. 4C). Similar results were observed with the analysis of endogenous RelA; U_L26 attenuated the translocation of endogenous NF-κB to the nucleus upon TNF-α treatment (Fig. 4D). Analysis of IκB levels indicated that U_L26 expression stabilized IκB after TNF-α treatment (Fig. 4E). Taken together, these results indicate that U_L26 expression is sufficient to stabilize IκB and block NF-κB activation after TNF-α treatment.

The HCMV U_L26 protein blocks NF-κB activity induced by Sendai virus infection.

To delineate whether U_L26-mediated inhibition of NF-κB activation was specific for TNF-α, we sought to test if U_L26 inhibited NF-κB activation in response to other NF-κB inducers. Toward this end, we infected cells with Sendai virus (SeV), which induces both NF-κB and IRF3 activation through a RIGI-dependent pathway (47) (Fig. 5A). SeV-infected cells exhibited pronounced NF-κB-dependent luciferase activity (Fig. 5B). This induction was substantially reduced by transfection with U_L26 (Fig. 5B). These results demonstrate that in addition to blocking TNF-α-induced NF-κB activation, expression of U_L26 blocks NF-κB activation mediated by an alternative pathway, indicating that U_L26 is acting at a conserved downstream point within these pathways.

SeV infection also resulted in a strong elevation of IRF3-dependent luciferase activity (Fig. 5C). This induction of IRF3 activity was unaffected by expression of U_L26 (Fig. 5C). U_L26 expression also had no effect on TNF-α-induced IRF3 nuclear translocation (Fig. 5D). These results suggest that U_L26-mediated inhibition of NF-κB signaling occurs downstream of the divergence between TNF-α's and SeV's stimulation of IRF3 and NF-κB signaling. Further supporting a lack of impact on IRF3 signaling, infection with a U_L26-deletion mutant did not impact IRF3 localization during HCMV infection (Fig. 5E).

The HCMV U_L26 protein blocks phosphorylation of the IKK complex.

Our findings that U_L26 blocks NF-κB activation induced by either TNF-α or SeV infection suggest that U_L26 blocks NF-κB activation downstream of where these pathways converge, which occurs at the activation of the IKK complex. In combination with the findings that U_L26 maintains IκB levels in the presence of TNF-α signaling (Fig. 4E), our results suggest that the U_L26 site of action is either IκB or the IKK complex. The IKK complex is active when IKKα/β is serine phosphorylated in its activation loop, which is mediated either through an upstream kinase or through autophosphorylation (48). We examined IKK phosphorylation in WT- or ΔU_L26-infected cells. At 48 hpi, in the absence of TNF-α, WT- and ΔU_L26-infected cells exhibited roughly equivalent amounts of IKK phosphorylation (Fig. 6A). The levels of IKK phosphorylation during infection with either virus were increased in comparison to levels seen with mock-infected cells, suggesting a higher level of basal phosphorylation during infection (Fig. 6A). At this same time point, upon TNF-α treatment, WT-infected cells exhibited similar amounts of IKK phosphorylation as untreated cells, consistent with an HCMV-induced block to TNF-α-induced IKK phosphorylation and activation. In contrast, mock- or ΔU_L26-infected cells showed an increase in IKK phosphorylation upon TNF-α treatment, consistent with IKK activation (Fig. 6A). These results suggest HCMV-mediated attenuation of TNF-α-induced IKK phosphorylation requires functional U_L26. It has been previously reported that HCMV blocks TNF-α-induced NF-κB signaling at 48 hpi, but not prior to 24 hpi (39). If such is the case, it would suggest that tegument protein delivery, which would include the U_L26 protein, is not sufficient to block TNF-α-induced NF-κB activation. To further explore this issue, we tested whether WT or UV-irradiated HCMV was sufficient to block TNF-α-induced IKK phosphorylation at various times postinfection. As shown in Fig. 6B, neither WT HCMV nor UV-HCMV was capable of blocking TNF-α-induced IKK phosphorylation at 6 hpi. At 24 hpi, TNF-α-induced IKK phosphorylation was slightly reduced in HCMV-infected cells relative to TNF-α-treated mock-infected cells (Fig. 6B). In contrast, at 48 hpi, WT HCMV infection, but not that with UV-irradiated HCMV, resulted in a complete block in IKK phosphorylation (Fig. 6B). These results suggest that HCMV-mediated tegument protein delivery is not sufficient to block TNF-α-induced IKK phosphorylation. To determine if U_L26 expression is sufficient to block TNF-α-induced IKK phosphorylation in the absence of other viral proteins, we transfected cells with U_L26, treated them with TNF-α, and assessed IKK phosphorylation. Expression of the U_L26 protein in the absence of other HCMV proteins blocked TNF-α-induced IKK phosphorylation (Fig. 6C). These results suggest that the U_L26 protein blocks TNF-α-induced IKK phosphorylation, thereby attenuating NF-κB signaling.