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Journal of Virology, December 2006, p. 11686-11698, Vol. 80, No. 23
0022-538X/06/$08.00+0     doi:10.1128/JVI.01168-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Human Cytomegalovirus Blocks Tumor Necrosis Factor Alpha- and Interleukin-1ß-Mediated NF-{kappa}B Signaling{triangledown}

Christina Montag, Jutta Wagner, Iris Gruska, and Christian Hagemeier*

Laboratory of Molecular Biology, Children's Hospital, Charité, Humboldt University Berlin, Berlin, Germany

Received 6 June 2006/ Accepted 12 September 2006


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ABSTRACT
 
NF-{kappa}B plays an important role in the early cellular response to pathogens by activating genes involved in inflammation, immune response, and cell proliferation and survival. NF-{kappa}B is also utilized by many viral pathogens, like human cytomegalovirus (HCMV), to activate their own gene expression programs, reflecting intricate roles for NF-{kappa}B in both antiviral defense mechanisms and viral physiology. Here we show that the NF-{kappa}B signaling pathway stimulated by proinflammatory cytokines tumor necrosis factor alpha (TNF-{alpha}) and interleukin-1ß (IL-1ß) becomes inhibited in HCMV-infected cells. The block to NF-{kappa}B signaling is first noticeable during the early phase of infection but is fully established only at later times. Biochemical and genetic evidence demonstrates that the viral inhibition of proinflammatory signaling by distinct cytokines occurs upstream of the convergence point of NF-{kappa}B-activating pathways, i.e., the I{kappa}B kinase complex, and that it is mediated via different mechanisms. Consistent with this, we further show that an HCMV variant that has lost the ability to downregulate TNF-{alpha}-induced NF-{kappa}B signaling also fails to downregulate surface expression of TNF receptor 1, thereby mechanistically linking the inhibition of TNF-{alpha}-induced NF-{kappa}B signaling by HCMV to TNF receptor targeting. Our data support a model whereby HCMV inhibits cytokine-induced NF-{kappa}B signaling at later times during infection, and we suggest that this contributes to the inhibition of the cell's antiviral defense program.


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INTRODUCTION
 
Nuclear factor {kappa}B (NF-{kappa}B) serves as an important cellular regulator of the earliest response to microbial pathogens. Activation of NF-{kappa}B is associated with the transcriptional induction of genes encoding chemokines, cytokines, adhesion molecules, proinflammatory enzymes, and proteins regulating apoptosis (26, 43). As such the transcriptional response mediated by NF-{kappa}B is an essential component both of an effective innate immune system and for mounting the adaptive immune response (10, 50).

In the classical pathway inactive NF-{kappa}B is retained in the cytoplasm by inhibitor of NF-{kappa}B (I{kappa}B) proteins, of which I{kappa}B{alpha} is the best-characterized member. Nuclear localization of NF-{kappa}B requires the proteasomal degradation of I{kappa}B. This step is tightly regulated and specifically induced upon I{kappa}B phosphorylation by the activated I{kappa}B kinase (IKK) complex. Once phosphorylated, I{kappa}B becomes polyubiquitinated and consecutively degraded by the proteasome (37). Therefore, the timely activation of the IKK is the single most important regulatory step in the induction of NF-{kappa}B transcriptional activity.

Several upstream pathways that feed into the classical NF-{kappa}B route have been identified, and they typically converge at the IKK. Of these, the best understood are the tumor necrosis factor (TNF) receptor 1 (TNFR1) and toll-like receptor (TLR)/interleukin-1 (IL-1) receptor (IL-1R) pathways. TNFR1 and TLR/IL-1 receptors signal to the IKK via distinct sets of adaptor proteins (29). TLRs and IL-1R very much rely on the same cytoplasmic pathway, but, whereas IL-1R binds the cytokine IL-1ß, TLRs are considered "pattern recognition receptors" that recognize and bind pathogen-associated microbial patterns like bacterial and viral constituents, lipopolysaccharides (LPS), and double-stranded RNA (34). Upon microbial encounter with cells TLRs elicit the first-line defense by activating the expression of type I interferons and other cytokines via an NF-{kappa}B-containing enhanceosome (38).

Viruses have developed not only numerous mechanisms to inhibit these antiviral response pathways as protective countermeasures but also strategies to integrate these activated cellular pathways into their own life cycles. For instance, viruses can inhibit interferon and cytokine production (40, 55) and function (4, 44, 57) and can also utilize activated NF-{kappa}B to kick-start viral early (E) gene expression programs (5, 31, 42, 49, 52). Furthermore, inflammatory cells that are attracted to the sites of infection as a consequence of pathway activation and chemokine production can be hijacked by viruses to support viral spread (27).

Human cytomegalovirus (HCMV), a member of the family Herpesviridae of double-stranded DNA viruses, activates NF-{kappa}B within minutes of virus-cell contact. At least in part this is brought about by TLR2 recognizing HCMV virions, resulting in the production of inflammatory cytokines in infected cells (18, 19), and the viral envelope glycoprotein gB appears to be necessary and sufficient for the instant activation of NF-{kappa}B (11, 61). These findings are further supported by microarray analyses of HCMV-infected cells showing that numerous interferon-stimulated genes and proinflammatory cytokines are upregulated immediately after infection, suggesting the instant activation of TLR/IL-1 and TNFR1 signaling in infected cells (12, 51, 63). Once activated, NF-{kappa}B can in turn stimulate viral immediate early (IE) gene expression (22, 46, 48). This step may also be of particular functional significance during the reactivation from latency (47). Thus, there appears to be an intricate virus-NF-{kappa}B interplay during the initial and immediate early phases of infection.

Interestingly, the microarray data also revealed that the activation of NF-{kappa}B-dependent genes was much stronger when HCMV gene expression was disallowed (12, 15), suggesting that virion factors elicit the activation of signaling pathways and that later viral gene products expressed at later times during infection have a negative influence on the NF-{kappa}B-dependent gene expression program. Consistent with this notion, HCMV has been reported to inhibit the transcription of the chemokine monocyte chemotactic protein 1 (MCP-1) (30), interferon response factor 3 (IRF3) (1), and the MxA gene (12) and to downregulate TNFR1 surface expression (6). Finally, the adapter kinase RICK has recently been shown to activate an NF-{kappa}B-dependent anti-HCMV response (23).

In this study we have undertaken a systematic analysis of the NF-{kappa}B signaling pathway during HCMV infection in the presence of proinflammatory cytokine stimulation. Our results show that during the initial phase of infection HCMV cannot counteract NF-{kappa}B activation by TNF-{alpha} and IL-1ß. However, HCMV can very effectively do so later during infection, suggesting the existence of one or more later viral gene products that can antagonize NF-{kappa}B signaling in infected cells. These results are consistent with a recent study by Jarvis and coworkers who also found that HCMV counteracts cytokine-induced NF-{kappa}B signaling (36). We identify an HCMV variant that fails to block NF-{kappa}B signaling after TNF-{alpha} stimulation but at the same time has maintained its ability to counteract IL-1ß-mediated NF-{kappa}B signaling. This variant helps to locate the block in NF-{kappa}B signaling upstream of the IKK and, for TNF-{alpha}-dependent stimulation, further suggests that the block to NF-{kappa}B signaling is causally linked to the HCMV-mediated downregulation of TNFR1 surface expression.


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MATERIALS AND METHODS
 
Cells, viruses, and proinflammatory induction. Human embryonic lung fibroblasts and U373 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 U of penicillin-streptomycin per ml. Cells were passaged 1:3 every 3 days. NF-{kappa}B was induced by stimulation with 30 ng/ml TNF-{alpha} (Sigma), 10 ng/ml IL-1ß (Biomol), or 1 µg/ml LPS (Sigma) for the times indicated in the legends for Fig. 1, 4, 5, and 7. Viruses were kindly provided by Hartmut Hengel, Bodo Plachter, and Susanna Proesch. The AD169 and Towne strains were purchased from ATCC. HEL fibroblasts or U373 cells at 70% confluence were infected with different HCMV strains at a multiplicity of infection (MOI) of 5 as indicated below. To monitor HCMV-dependent regulation of NF-{kappa}B activity, cells were infected with HCMV for various lengths of time (2 to 72 h) prior to the period of incubation with TNF-{alpha}, IL-1ß, or LPS as specified below. To inhibit viral late gene expression, cells were infected in the presence of 100 µM ganciclovir (GCV) (Sigma). Complete viral gene expression was inhibited by UV radiation (5,000 J/m2).


Figure 1
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  		FIG. 1. Inhibition of TNF-{alpha}-induced NF-{kappa}B DNA binding by HCMV. (A) Human primary fibroblasts were infected with HCMV AD169 (ATCC) at a MOI of 5 for 1 h and/or treated with 30 ng/ml TNF-{alpha} for 10 min. Then cells were harvested for the preparation of nuclear extracts. DNA binding activity of NF-{kappa}B (black arrow) was analyzed by EMSA using a specific or a mutated probe from the MHC I promoter as indicated. An antibody recognizing the p65 subunit of DNA-bound NF-{kappa}B was added to the reaction mixture, resulting in a "supershift" of the NF-{kappa}B complex (open arrow). *, nonspecific band. (B) Schematic of the experimental setup. Human primary fibroblasts were infected with HCMV as described above but for various lengths of time (between 0 and 72 h) as indicated by the bars. Ten minutes prior to harvest cells were stimulated with TNF-{alpha}. (C) Cells were infected for the indicated periods of time and stimulated with TNF-{alpha} for 10 min as described for panel B. Nuclear extracts were analyzed by EMSA. NF-{kappa}B DNA binding (arrow) was visualized by autoradiography. *, nonspecific band; FP, unbound (free) probe.


Figure 4
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  		FIG. 4. HCMV inhibits Il-1ß- and LPS-induced NF-{kappa}B activation. (A) Human primary fibroblasts were infected with AD169 (ATCC) and stimulated with IL-1ß for 30 min prior to harvesting. Nuclear extracts were prepared for EMSA to analyze NF-{kappa}B DNA binding. (B) Cells were treated as described for panel A with the exception of a 2-h rather than a 30-min incubation period with IL-1ß to allow sufficient RNA accumulation. Total RNAs were analyzed by multiprobe RPA for the relative abundance of the indicated NF-{kappa}B target gene transcripts. L32 was included to control for equal amounts of RNA loading per analysis. (C and D) For these experiments U373 cells were used since they can be well stimulated by LPS and are also permissive for infection with HCMV. LPS stimulation was initiated 1 h (C) or 2 h (D) prior to harvest of infected cells as indicated. In principle, EMSA (C) and RPA analyses (D) were performed as described for panels A and B, respectively.


Figure 5
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  		FIG. 5. Inhibition of NF-{kappa}B after TNF-{alpha} stimulation and inhibition after IL-1ß stimulation are separable functions of HCMV. Cells were infected with the HCMV variant AD169var-T as indicated and stimulated for either 10 min with TNF-{alpha} (A and C) or 30 min with IL-1ß (B and D) prior to harvesting and according to the schedule shown in Fig. 1B. Nuclear extracts were then prepared and NF-{kappa}B DNA binding activity analyzed by EMSA in principle as described for Fig. 3. (C and D) Immunoblot analysis of I{kappa}B{alpha} abundance in cells infected with HCMV for the indicated periods of time. An antibody against vigilin was used to control for equal protein loading. (E) Viral DNA from AD169 (ATCC), AD169var-T, and Towne was digested with EcoRI or ClaI, and the restriction pattern was visualized by ethidium bromide staining. The restriction pattern of Towne is more distinct from those of AD169 (ATCC) and AD169var-T, which in turn are very similar to each other. Obvious differences between the last two are indicated by an asterisk. (F) Cells were infected with AD169 (ATCC) or AD169var-T as indicated, and protein extracts were prepared. Expression of viral proteins representing the kinetic classes of IE (IE1 and IE2), E (p52), and L (gB and pp65) genes is shown by immunoblotting. A vigilin antibody was used to control for equal protein loading.


Figure 7
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  		FIG. 7. Viral gene expression but not expression of late genes is necessary for inhibition of NF-{kappa}B signaling and TNFR1 surface expression. Human embryonic lung cells were either infected with untreated and UV-inactivated AD169 (ATCC) for 72 h (A and B) or with AD169 (ATTC) in the presence and absence of GCV for various lengths of time as indicated (C and D). Prior to harvesting, cells to be used for immunoblot and EMSA analysis (A and C) were stimulated with 30 ng/ml TNF-{alpha} for 10 min where indicated. Antibodies used were directed against IE1/2 and I{kappa}B{alpha} (A) or against a late glycoprotein (gB) and I{kappa}B{alpha} (C). In both cases detection of vigilin was used to control for loading. EMSAs were performed as described for Fig. 1. Only the retarded NF-{kappa}B-DNA complexes are shown. (B and D) For the detection of TNFR1 surface expression cells were stained with PE- or FITC-conjugated antibodies as indicated and analyzed by FACS as described for Fig. 6.

Antibodies. P65 (C20), I{kappa}B{alpha} (C15), and IKK{gamma} (FL-419) antibodies were purchased form Santa Cruz, and the IKKß antibody was obtained from Invitrogen. gB, pp65, and p52 antibodies were purchased from Abi, and the IE1/IE2 antibody was received from Argene. For fluorescence-activated cell sorting (FACS) analysis phycoerythrin (PE)-conjugated TNFR1 and fluorescein isothiocyanate (FITC)-conjugated CD95 were used (R&D Systems). PE- and FITC-labeled isotype controls were also purchased from R&D Systems.

Immunoblotting. Cells were lysed in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol (DTT), 10 mM NaF, 80 mM ß-glycerolphosphate, 100 µM NaVO4, 0.4 µM Pefabloc, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin for 1 hour. Lysates were centrifuged for 10 min, and the protein concentration of clear supernatants was measured using the Bradford assay (Bio-Rad). Equal amounts of proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoreses and transferred to a polyvinylidene difluoride membrane by semidry blotting. After being blocked in 5% milk powder-phosphate-buffered saline-Tween 20 (PBS-T) for 1 h, membranes were exposed to antibody solution (5% milk powder-PBS-T) for 1.5 h at room temperature or overnight at 4°C. Generally these and other experiments were performed at least three times, and representative results were used for preparing figures.

Coimmunoprecipitation and kinase assays. Cells were lysed as described for immunoblotting. One hundred micrograms of protein extracts was first precleared with protein A-Sepharose beads for 1 h at 4°C. Immunoprecipitation using 1 µg IKK{gamma} antibody was carried out for 2 h at 4°C. Protein A-Sepharose was washed five times with lysis buffer and once with kinase reaction buffer containing 20 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM ß-glycerolphosphate, 50 µM NaVO4, 1 mM DTT, and 20 µM ATP. Kinase reactions were performed with 5 µCi of [{gamma}-32P]ATP and 1 µg of glutathione S-transferase (GST)-I{kappa}B{alpha}1-53 (kindly provided by Claus Scheidereit, Max Delbrück Center for Molecular Medicine) as the substrate in 15 µl for 20 min at 37°C. After addition of SDS loading buffer, reaction mixtures were boiled and applied to an SDS-polyacrylamide gel. The dried gel was analyzed by autoradiography. Experiments were done at least three times, with typical results shown.

EMSA. Cells were incubated in hypotonic buffer (10 mM HEPES, pH 7.5, 10 mM KaCl, 3 mM MgCl2, 0.05% NP-40, 1 mM EDTA, pH 8.0, 10 mM NaF, 0.1 mM NaVO4, 1 mM Pefabloc, 1 mM DTT, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) for 2 min and centrifuged for 2 min at 1,000 x g. Pellets were washed twice with hypotonic buffer and lysed in 250 µl lysis buffer (0.5 M KaCl, 35% glycerol, 100 mM HEPES, pH 7.4, 5 mM MgCl2, 0.5 mM EDTA, pH 8.0, 5 mM NaF, 1 mM Pefabloc, 1 mM DTT, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). Lysates were centrifuged again, resuspended in lysis buffer, and centrifuged for 45 min at 4°C at 100,000 x g, and the protein concentration of clear supernatants (nuclear extract) was measured using the Bradford assay (Bio-Rad). An NF-{kappa}B-specific oligonucleotide (5'-GAT CCA GGG CTG GGG ATT CCC CAT CTC CAC AGG-3' or 5'-GAT CCC TGT GGA GAT GGG GAA TCC CCA GCC CTG-3') or a mutated oligonucleotide (5'-GAT CCA GGG CTG GGC ATT CCG CAT CTC CAC AGG-3' or 5'-GAT CCC TGT GGA GAT GCG GAA TGC CCA GCC CTG-3') from the H-2K region of the major histocompatibility complex class I (MHC-I) promoter was labeled with [{alpha}-32P]ATP in the presence of 500 mM deoxynucleoside triphosphates without ATP for 30 min at 37°C and for 5 min at 65°C. Nonincorporated radioactivity was removed by NICK Sephadex G-50 columns (Amersham). For the binding reaction 5 µg of nuclear extracts was incubated with 2x shift buffer (40 mM HEPES, pH 7.9, 120 mM KCl, 8% Ficoll), 0.5 µg/µl bovine serum albumin, 5 nM DTT, 0.5 µg/µl poly(dIdC), and 20,000 cpm of labeled H2-K oligonucleotide for 30 min at 30°C. Supershift assays were carried out by preincubating the reaction mixture with 2 µg of an anti-p65 antibody or control for 30 min at 4°C. DNA binding was analyzed on 5% polyacrylamide gels by autoradiography. Electrophoretic mobility shift assays (EMSAs) were performed at least three times, and representative results were used for preparing figures.

RPA. RNase protection assays (RPA) were performed essentially as described previously (60). Briefly, multiprobe RNase protection assays were performed by using the Riboquant system (Pharmingen). Defined pools of [32P]UTP-labeled probes were synthesized by in vitro transcription from a custom-made template set (Pharmingen). Total RNAs from cells were isolated using Tri-Reagent (Invitrogen) by following the manufacturer's instructions. One microgram of each preparation was used as target RNA in the hybridization reaction. Then RNA samples were RNase treated, purified, and subjected to denaturing polyacrylamide gel electrophoresis followed by autoradiography and quantitation by phosphorimaging. Experiments were performed at least three times, with representative results used for preparing figures.

Restriction endonuclease digestion. Human embryonic lung fibroblasts were infected at a MOI of 5. After 5 days of infection cells were frozen at –80°C and thawed at 37°C. Supernatants were collected and centrifuged at 9,000 x g for 10 min at 4°C, and the pellet was than incubated in a RNase/DNase solution (100 µg/ml RNase (Sigma) and 100 U/ml DNase (Sigma) for 15 min at 37°C. The supernatant was centrifuged at 50,000 x g for 1 h by 4°C. The resulting pellet was resuspended in an SDS-proteinase K solution (10% SDS, 500 ng/ml proteinase K; Sigma) for 2 h at 55°C. DNA was purified by phenol-chloroform precipitation and dissolved in H2O. For restriction endonuclease digestions 2 µg viral DNA was incubated with 4 units of enzymes for 3 h at 37°C. The digestion products were separated in a 0.8% agarose gel, followed by ethidium bromide staining.

FACS. Cells (105) were incubated in 25 µl PBS containing 0.5% bovine serum albumin and 2 ng/µl of either a PE-conjugated TNFR1 antibody or a PE-conjugated isotype control for 30 min at 4°C in the dark. The CD95 antibody was FITC labeled. Cells were washed twice in PBS and measured in the FL-2 (PE) or the FL-1 (FITC) channel of a FACScan (Becton Dickinson).


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RESULTS
 
HCMV inhibits TNF-{alpha}-mediated NF-{kappa}B DNA binding activity. Whereas most studies analyzing the regulation of NF-{kappa}B by HCMV have concentrated on early events elicited by viral infection, the present work aimed at studying the integrity of the NF-{kappa}B signaling pathway in infected cells that were stimulated with NF-{kappa}B-inducing agents like TNF-{alpha}. By so doing, we focused on identifying possible viral modulatory events affecting the host cell's ability to activate the NF-{kappa}B signaling pathway. Treatment of cells with 30 ng/ml TNF-{alpha} for 10 min leads to the well-recognized activation of NF-{kappa}B DNA-binding activity, as shown by an EMSA using nuclear extracts and an NF-{kappa}B binding site derived from the MHC-I promoter (Fig. 1A, lane 4). This binding is specific since a probe containing a mutated NF-{kappa}B binding site is not retarded (lane 5). As expected, infection of primary fibroblasts with HCMV AD169 (ATCC) at a MOI of 5 for 1 h also resulted in a specific NF-{kappa}B-DNA complex (lanes 7 and 8). TNF-{alpha} treatment and HCMV infection had no significant additional effect on the cells' NF-{kappa}B pool to bind DNA (lanes 10 and 11). When a specific antibody directed towards the NF-{kappa}B subunit p65 (RelA) was added to the reaction mixture, the relevant DNA-protein complexes were "supershifted" (lanes 3, 6, 9, and 12). This was not the case when a nonspecific control antibody was used (data not shown). These data suggest that p65 is a constituent of the DNA-protein complexes detected in TNF-{alpha}-stimulated and HCMV-infected cells, indicating the induction of the canonical or "classical" NF-{kappa}B pathway.

In order to analyze whether HCMV can modulate the induction of NF-{kappa}B signaling during the course of infection, we next set up the experimental system that is schematically shown in Fig. 1B. Accordingly, fibroblasts were infected for various lengths of time (0, 2, 4, 6, 8, 10, 24, 34, 48, 58, or 72 h) with HCMV AD169 at a MOI of 5. This infectious period is referred to as "preinfection". After preinfection, cells were treated with TNF-{alpha} (30 ng/ml) for 10 min. Subsequently, cells were harvested, nuclear extracts were prepared, and NF-{kappa}B DNA binding was analyzed by EMSA. TNF-{alpha} treatment alone results in a marked upregulation of NF-{kappa}B DNA binding (Fig. 1C, lane 2). Preinfection with HCMV for up to 6 h prior to TNF-{alpha} treatment had no significant effect on TNF-{alpha}-induced NF-{kappa}B DNA binding. Under conditions employing preinfection times of 8 and 24 h TNF-{alpha}-induced NF-{kappa}B binding was slightly reduced, and preinfection for 34 h or longer entirely counteracted TNF-{alpha}-mediated activation of NF-{kappa}B. These data suggest that during the initial time of an HCMV infection the virus cannot antagonize NF-{kappa}B activation by TNF-{alpha}. However, by around 34 h after preinfection initiation, cells have gained an additional function which renders these cells unresponsive to TNF-{alpha}-mediated activation of NF-{kappa}B.

HCMV inhibits TNF-{alpha}-induced NF-{kappa}B target gene expression and signaling. TNF-{alpha} binds to and activates TNFR1. TNFR1 signals to NF-{kappa}B via several well-recognized adaptor proteins (3, 32, 62), and NF-{kappa}B regulates numerous genes involved in mounting the host's immune response to various pathogens and genes with important functions in regulating the differentiation, proliferation, and apoptotic programs of cells (7). In order to follow TNF-{alpha} signaling in infected cells, we first analyzed the transcriptional induction of a number of well-defined NF-{kappa}B target genes, namely, the TRAF1, I{kappa}B{alpha}, and cIAP2 genes, by RNase protection assay. All three target genes were found to be strongly induced after stimulation with TNF-{alpha} in the absence of HCMV infection (Fig. 2A, lane 2, and B). However, if cells were preinfected with HCMV prior to TNF-{alpha} treatment following the schedule schematically shown in Fig. 1B, induction of NF-{kappa}B target genes was attenuated over the time of the preinfection. Although we have not formerly excluded the possibility that the decrease in steady-state mRNA levels could be due to an increase in mRNA decay, the congruent regulation of the NF-{kappa}B DNA binding activity (Fig. 1C) and the specific regulation of the more upstream parts of the NF-{kappa}B signaling pathway (see below) strongly suggest that the decline in mRNAs observed here is primarily the result of a reduced level of transcription of these genes. This suggested that the accumulation of viral gene products counteracted TNF-{alpha}-induced NF-{kappa}B target gene activation (Fig. 2A, lanes 3 to 7, and B). The fact that not all NF-{kappa}B target genes were affected in an identical manner suggests that NF-{kappa}B-independent regulatory mechanisms also influence expression of these genes in infected cells. Still, genes that are not NF-{kappa}B target genes like the caspase 8 and TNFR2 genes were not affected by TNF-{alpha} treatment, and TNFR2 gene expression was even slightly upregulated at late times of infection as previously reported (12). As expected, from the reduction in mRNA levels the inhibitory effect of HCMV on TNF-{alpha} signaling could also be observed at the protein level for NF-{kappa}B target genes (data not shown).


Figure 2
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  		FIG. 2. HCMV inhibits TNF-{alpha}-induced NF-{kappa}B target gene expression. (A) Human primary fibroblasts were HCMV infected as described for Fig. 1 for the indicated time periods and treated with TNF-{alpha} for 2 h. Total RNAs were prepared, and a multiple-probe RPA was performed. A set of probes including NF-{kappa}B target genes encoding Traf1, I{kappa}B{alpha}, and cIAP2 was used. L32 was included to control for RNA quality and equal loading. (B) The RPA analysis shown in panel A was quantified using a phosphorimager (Fuji).

Given the inability of TNF-{alpha} to induce NF-{kappa}B target genes in cells that were preinfected for 24 h or longer (Fig. 2), we reasoned that HCMV is likely to inhibit NF-{kappa}B signaling later during infection. Therefore, we next analyzed the NF-{kappa}B signaling pathway in cells that were HCMV infected for different time periods and then TNF-{alpha} treated according to the schedule shown in Fig. 1B. NF-{kappa}B is kept under tight control by I{kappa}B{alpha}, which normally retains this transcription factor in the cytoplasm. In the case of NF-{kappa}B pathway activation I{kappa}B{alpha} first becomes phosphorylated by the IKK and is subsequently ubiquitinated and degraded in a proteasome-dependent manner, which facilitates nuclear entry and DNA binding of NF-{kappa}B (37). As shown before, a 10-min stimulation of cells with TNF-{alpha} leads to a strong induction of NF-{kappa}B DNA binding activity (Fig. 3A, lane 2), and this is consistent with the rapid loss of I{kappa}B{alpha} (Fig. 3B, lane 2) and activation of the IKK (Fig. 3C, lane 2). If TNF-{alpha} is added after a preinfection period of 4 h, the situation is very much unchanged, with IKK activity, I{kappa}B{alpha} instability, and NF-{kappa}B DNA binding activity still induced (Fig. 3A to C, lanes 3). However, if preinfection is allowed to proceed for 24 to 72 hpi before TNF-{alpha} was added, dramatic differences in NF-{kappa}B signaling in HCMV-infected cells occur. TNF-{alpha}-induced IKK activity is first hampered and then completely abrogated in these cells (Fig. 3C, lanes 4 to 6). Consistent with the downregulation of IKK activity, I{kappa}B{alpha} degradation is inhibited in these cells (Fig. 3B, lanes 4 to 6) and DNA binding of NF-{kappa}B is first significantly reduced (Fig. 3A, lane 4) and finally inhibited (lanes 5 and 6). These data are in line with the expression analysis of TNF-{alpha}-inducible genes in HCMV-infected cells (Fig. 2), and together they show that during the initial phase of an infection with HCMV TNF-{alpha}-induced NF-{kappa}B signaling is still in place. However, later viral gene expression first attenuates and then shuts down this signaling pathway.


Figure 3
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  		FIG. 3. Inhibition of NF-{kappa}B signaling by HCMV. Cells were infected with different HCMV strains and clinical isolates under similar conditions (MOI, 5) as indicated and treated with TNF-{alpha} for 10 min according to the schedule shown in Fig. 1B. After being harvested, cells were used to prepare extracts for analyzing NF-{kappa}B signaling at various levels of the pathway including NF-{kappa}B DNA binding, stability of I{kappa}B{alpha}, and IKK activity. (A) EMSA for NF-{kappa}B with extracts prepared from cells infected with the HCMV laboratory strain AD169 (ATCC). Because of space limitations only the retarded NF-{kappa}B-DNA complex is shown. This complex is specific and can be "supershifted" with an antibody recognizing the p65 subunit of DNA-bound NF-{kappa}B as shown in Fig. 1A (data not shown). (B) Immunoblot demonstrating the protein stability of I{kappa}B{alpha} in AD169-infected and TNF-{alpha}-stimulated cells. An antibody against vigilin was used to control for protein loading. (C) In vitro kinase assay following the immunoprecipitation of the IKK complex from AD169-infected and TNF-{alpha}-stimulated cells by an antibody directed against the IKK{gamma} subunit as indicated. Purified GST-I{kappa}B{alpha} served as a substrate for the IKK. IKKß was analyzed by immunoblotting to control for the relative abundance of precipitated kinase complexes. (D to F) EMSA for NF-{kappa}B DNA binding. Only the retarded complexes are shown. Experiments were performed as described for panel A with the exception that cells were infected either with the laboratory strain Towne (ATCC) (D) or low-passage clinical isolates LPCI-I (E) and LPCI-II (F).

For the experiments so far we have used the HCMV AD169 (ATCC) laboratory strain, and the experiments were reproducibly shown using both different virus preparations and also purified virus (data not shown). To further exclude strain-specific effects, we next used an independent laboratory strain, Towne (ATCC), and two low-passage HCMV clinical isolates (kindly provided by the late S. Prösch, Berlin) and asked whether the above results could be reproduced under these conditions. Like AD169, both the Towne strain and the two low-passage clinical isolates showed a strong inhibition of TNF-{alpha}-induced NF-{kappa}B DNA binding activity. Whereas preinfection with the Towne strain for 48 h resulted in an entire block of NF-{kappa}B activation (Fig. 3D), a 48-h preinfection of cells with the clinical isolates attenuated NF-{kappa}B activation to a great extent, albeit not completely (Fig. 3E and F). These data support the previous results and, in addition, show that the inhibitory activity of HCMV directed against TNF-{alpha}-induced NF-{kappa}B activation is strain independent.

HCMV inhibits Il-1ß- and LPS-stimulated NF-{kappa}B signaling. The data shown in Fig. 3C suggest that HCMV inhibits TNF-{alpha}-induced NF-{kappa}B signaling at or upstream of the IKK. Since the IKK is the convergence point for several distinct pathways that signal from the cell surface to NF-{kappa}B (29), we wondered whether HCMV could also counteract the activation of NF-{kappa}B after stimulation of TNFR1-independent pathways. We concentrated on IL-1ß, another important proinflammatory cytokine during viral infection that induces a similar set of target genes like the TNF-{alpha} gene. IL-1ß binds to the IL-1 receptor and signals to the IKK by means of adapter molecules (2, 13, 35) that to a large extent are distinct from those employed by TNFR1. After stimulation with 10 ng/ml IL-1ß for 30 min NF-{kappa}B DNA binding activity is strongly induced (Fig. 4A, lane 2), and preinfection of cells with HCMV for up to 10 h prior to IL-1ß treatment had little effect on these findings (lanes 3 and 4). However, preinfection for 24 h or more led to a strong inhibition of the DNA binding activity of NF-{kappa}B (lanes 5 to 7). The increase in NF-{kappa}B DNA binding is consistent with the induction of NF-{kappa}B target genes (Fig. 4B, lanes 2 to 4), but, importantly, these target genes remained noninduced when cells were preinfected with HCMV for 24 h or longer despite IL-1ß treatment (lanes 5 to 7). These data show that HCMV can block NF-{kappa}B activation after stimulation by a different proinflammatory cytokine that utilizes distinct signaling pathways upstream of the IKK.

This view is further supported by our finding that HCMV also counteracts NF-{kappa}B signaling after stimulation of cells with 1 µg/ml LPS for 1 h (Fig. 4C and D). It is considered that IL-1ß and LPS depend to a large extent on the same intracellular signaling pathway upstream of the IKK, with the obvious exception that LPS activates the pattern recognition receptor TLR4 (9). The differences in the kinetics and strength of the HCMV-mediated block in NF-{kappa}B activation between IL-1ß- and LPS-stimulated cells are likely due to the fact that we had to use the glioblastoma cell line U373 for LPS stimulation, since, unlike U373 cells, primary fibroblasts cannot efficiently be stimulated by LPS. Although we cannot exclude the possibility that the small differences we observed point to meaningful differences, the more important conclusion from this set of experiments is that HCMV can block NF-{kappa}B activation following the stimulation of three distinct surface receptors.

Downregulation of NF-{kappa}B after TNF-{alpha} stimulation and downregulation after IL-1ß stimulation are separable functions of HCMV. As shown in Fig. 3 the ability to block NF-{kappa}B activation after proinflammatory stimulation was a strain-independent activity of HCMV. On the other hand, this anti-NF-{kappa}B activity is unlikely to be an essential component of the viral life cycle in cultured cells and, if so, there should be little selective pressure on maintaining this viral function. Therefore, we considered it possible that the anti-NF-{kappa}B activity might have been lost from some of the high-passage laboratory strains of HCMV (for an example, see reference [20]) and screened different strains, intrastrain variants, and low-passage clinical isolates at similar MOIs for their abilities to block NF-{kappa}B activity after proinflammatory cytokine stimulation. We were unable to identify an HCMV strain/variant that had lost the ability to counteract NF-{kappa}B activity after IL-1ß stimulation (data not shown). However, we did identify an AD169 strain of HCMV that had completely lost the ability to block NF-{kappa}B signaling after TNF-{alpha} stimulation (Fig. 5A). We refer to this strain as AD169var-T. As implied above, AD169var-T could still counteract NF-{kappa}B activation after IL-1ß treatment (Fig. 5B) or following LPS stimulation (data not shown). Consistent with the inability of AD169var-T to counteract NF-{kappa}B DNA binding after TNF-{alpha} stimulation, I{kappa}B{alpha} remained unstable in these cells even if stimulation followed a 72-h preinfection period (Fig. 5C). In contrast, after IL-1ß stimulation I{kappa}B{alpha} was stable when stimulation occurred in cells preinfected for at least 48 h (Fig. 5D).

A comparative restriction enzyme analysis of AD169 (ATCC), Towne (ATCC), and AD169var-T demonstrated that AD169var-T is indeed very similar but not identical to AD169 (ATCC) and that the two AD169 strains are more closely related to each other than to the Towne strain (Fig. 5E). On this basis and additional restriction analyses with different enzymes showing similar results (data not shown) it seems reasonable to denominate AD169var-T as an AD169 variant (45). Also, it seems unlikely that the observed differences in counteracting TNF-{alpha}-induced NF-{kappa}B signaling are simply the consequence of differences in viral gene expression kinetics. As seen from Fig. 5F both AD169 (ATCC) and AD169var-T express IE, E, and late (L) genes with similar kinetics and levels of strength. Thus, these data show that blocking NF-{kappa}B activation following TNF-{alpha} stimulation and blocking activation after IL-1ß stimulation are separable viral functions, implying that distinct viral gene products mediate these effects.

AD169var-T mechanistically links the block of NF-{kappa}B activation to the downregulation of TNFR1 surface expression. Since AD169var-T discriminates between the inhibition of NF-{kappa}B after stimulation with TNF-{alpha} and IL-1ß, the above results also support the view that the HCMV-mediated blockages to NF-{kappa}B activation occur upstream of the convergence point (IKK), i.e., in the noncommon branches of the TNF-{alpha} and IL-1ß signaling pathways. This notion is consistent with the HCMV-mediated block of IKK activation after TNF-{alpha} treatment (Fig. 3C). On this basis two predictions can be made with respect to the mechanisms employed by HCMV: (i) the mechanism underlying the block of TNF-{alpha}-stimulated NF-{kappa}B activation should affect pathway components that are located between the IKK and TNFR1 and (ii) any functionally significant modification to this part of the pathway should be present in cells infected with HCMV AD169 (ATCC) and Towne (ATCC) but absent from cells infected with AD169var-T. To address these points, we looked for the respective pathway components in cells infected with AD169 (ATCC), Towne (ATCC), or AD169var-T. Since the activation of the IKK is still poorly understood, we first concentrated on analyzing the relative abundance of receptor-proximal components of the TNF-{alpha} signaling pathway but found no differences in the relative expression levels of TRADD, TRAF2, or RIP (data not shown). We then analyzed the surface expression of TNFR1, which had recently been reported to be negatively regulated by HCMV (6). Consistent with this, surface expression of the TNFR1 becomes downregulated in AD169 (ATCC)-infected cells (Fig. 6, row 1 [top]) and Towne (ATCC)-infected cells behave in an indistinguishable manner (Fig. 6, row 2). This effect is specific since the surface expression of the unrelated CD95 receptor was not affected by AD169 (ATCC) (Fig. 6, row 3), and mock-infected cells showed no loss of the TNFR1 receptor (Fig. 6). However, in sharp contrast, AD169var-T was unable to inhibit the surface expression of TNFR1 (Fig. 6, row 4), and this is consistent with the sustained induction of TNF-{alpha}-mediated NF-{kappa}B DNA binding activity (Fig. 5A) and instability of I{kappa}B{alpha} (Fig. 5B) in AD169var-T-infected cells. Thus, AD169var-T allows mechanistic linkage of the block of NF-{kappa}B activation after proinflammatory stimulation by TNF-{alpha} to the HCMV-induced downregulation of TNFR1.


Figure 6
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  		FIG. 6. AD169var-T links the block of NF-{kappa}B activation in HCMV-infected cells to TNFR1 downregulation. Primary human fibroblasts were infected with HCMV AD169, Towne, and AD169var-T as indicated. Cells were harvested and incubated with either PE- or FITC-conjugated antibodies (dark line) directed against TNFR1 or CD95 or their respective isotype controls (thin line). Cells were then analyzed by FACS (Becton Dickinson) for the surface expression of TNFR1 and CD95 during the course of an infection with HCMV as indicated.

An early gene product appears to be responsible for counteracting both TNF-{alpha}-induced NF-{kappa}B signaling and TNFR1 surface expression. The previous results have shown that counteracting TNF-{alpha}-induced NF-{kappa}B signaling is detectable only between 6 to 8 h postinfection and is not fully in place before 24 hpi. In order to start analyzing which viral gene(s) might be responsible for the observed effects, we addressed the question of which kinetic class of viral genes might cause the observed effects. We first asked whether viral gene expression is required at all or whether, for instance, tegument proteins could be responsible for the NF-{kappa}B-counteracting activities. To this end, we treated virus with UV prior to infection. As expected UV treatment abrogates viral IE gene expression (Fig. 7A, lane 4), which in cells infected with untreated virus is easily detectable 72 hpi (lane 3). As seen before, TNF-{alpha}-induced I{kappa}B{alpha} degradation and NF-{kappa}B DNA binding activity are efficiently counteracted in cells preinfected for 72 h (lane 3). In contrast, UV-treated HCMV lost the capability to inhibit NF-{kappa}B signaling and DNA binding (lane 4), indicating that viral gene expression is required for this viral activity. Also, downregulation of TNFR1 is dependent on viral gene expression (Fig. 7B). We then asked whether viral DNA replication, i.e., true late gene expression, is required for the NF-{kappa}B-inhibitory activity. Therefore, we analyzed NF-{kappa}B DNA binding and the abundance of I{kappa}B{alpha} in the absence or presence of GCV, an inhibitor of HCMV DNA replication. Figure 7C shows that GCV treatment effectively abrogates late gene (gB gene) expression, indicating that the block to viral late gene expression is well in place. Irrespective of the presence of GCV I{kappa}B{alpha} degradation and NF-{kappa}B DNA binding after TNF-{alpha} treatment are inhibited by the virus (Fig. 7C). However, we have also observed that under GCV treatment I{kappa}B{alpha} levels are not fully restored (compared to untreated cells) and NF-{kappa}B DNA binding is not completely inhibited (Fig. 7C). These results indicate that under our experimental conditions viral true late genes are not strictly required to counteract NF-{kappa}B signaling after TNF-{alpha} treatment. Thus, a viral gene(s) expressed with early kinetics is able to counteract TNF-{alpha}-induced NF-{kappa}B signaling. Still, an additional gene product expressed with late kinetics appears to be required for the full inhibitory activity to unfold. Inhibition of TNFR1 surface expression by the virus is clearly independent of GCV treatment (Fig. 7D), again suggesting that an early gene product is responsible for the downregulation. Interestingly, and in contrast to the inhibition of the NF-{kappa}B signaling shown above, early gene products seem to be sufficient for mediating this effect.


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DISCUSSION
 
Here we report the inhibition of NF-{kappa}B activation by HCMV after proinflammatory stimulation, which is a new property of this virus. It affects TNFR1-, IL-1R-, and TLR-mediated signal transduction and results in a block of NF-{kappa}B-mediated target gene expression at later times during infection.

The regulation of NF-{kappa}B in HCMV-infected cells has been a matter of intense research over recent years, but most studies so far have concentrated on the direct and mutual activation of HCMV gene expression and cellular NF-{kappa}B signaling. NF-{kappa}B is strongly induced right at the beginning of the infection, which is mainly caused by the physical interaction of viral surface constituents with cellular TLRs (11, 19, 53, 61). This occurs at times of viral attachment and entry via integrins (24, 58) and the epidermal growth factor receptor (EGFR) (59). Consistent with this view, the same viral glycoprotein (gB) has been shown to interact with TLR2, EGFR, and integrin {alpha}vß3 (39, 44, 59), suggesting that by default viral entry is linked to the early induction of the cellular innate immune response, of which the activation of the TNF-{alpha} and IL-1ß signaling pathways is a critical hallmark (34). The work presented here, demonstrating the ability of HCMV to downregulate TNF-{alpha}- and IL-1ß-induced NF-{kappa}B signaling, is in line with recent publications suggesting that HCMV has evolved mechanisms to counteract this cellular innate immune response. Previously, gene expression and microarray analyses have revealed that the expression of interferon response genes and proinflammatory cytokines was much increased when viral gene expression was inhibited, suggesting that one or more HCMV proteins function to block the expression of these genes during infection (12, 15, 30). The data from this study together with a recent publication by Jarvis and coworkers (36) now show more directly that HCMV can indeed actively block induction of NF-{kappa}B signaling after proinflammatory stimulation of infected cells by TNF-{alpha} and IL-1ß. In agreement with the study by Jarvis et al. (36) we show that NF-{kappa}B target gene expression and DNA binding activity as well as I{kappa}B{alpha} stability following proinflammatory stimulation are counteracted by HCMV. In addition to Jarvis et al. we directly show that the IKK is also inhibited by HCMV (Fig. 3), and the identification of AD169var-T further adds genetic evidence (i) that NF-{kappa}B inhibition through TNF-{alpha} and inhibition through IL-1ß are (genetically) separable activities of HCMV and (ii) that the level of inhibition must be located upstream of the convergence point (the IKK) of the NF-{kappa}B activating pathways. Consistent with this conclusion we show that the previously demonstrated ability of HCMV to downregulate TNFR1 surface expression (6), which we also observed after infection with the two clinical isolates presented in Fig. 3 (data not shown), is specifically lost in AD169var-T (Fig. 6). The mutual regulation of NF-{kappa}B and HCMV is summarized in Fig. 8.


Figure 8
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  		FIG. 8. Mutual regulation of HCMV and NF-{kappa}B. NF-{kappa}B is activated by HCMV-cell contacts (A). NF-{kappa}B in turn induces the gene expression program that initiates the innate (and adaptive) immune response (B) to combat viral infection (C). In addition, NF-{kappa}B costimulates viral IE gene expression (D), which is switched on by a plethora of cellular transcription factors (and possibly additional viral proteins) that are either constitutively expressed or, much like NF-{kappa}B, induced by virus-cell contacts (e.g., via EGFR signaling) (E). There is also evidence for viral IE proteins activating the expression of NF-{kappa}B subunit genes (F). Viral IE proteins promote later viral gene expression (G), and these genes encode several factors that realize the viral immune evasion program (H). Alike, later viral gene products block NF-{kappa}B activation after proinflammatory stimulation (present work), thereby contributing to counteracting the host's immune response program (I).

Our data strongly suggest that HCMV employs different gene products to block NF-{kappa}B activation through TNFR1 and TLR/IL-1R signaling. This is based on the observation that the HCMV variant AD169var-T has lost the ability to block NF-{kappa}B activation after TNF-{alpha} stimulation but can still prevent it following IL-1ß (Fig. 5) and LPS (data not shown) signaling. The block to TNF-{alpha}-induced NF-{kappa}B signaling was first observed between 6 and 24 hpi and complete after 34 hpi. This suggests that the responsible viral factors are expressed with E or L gene kinetics. Recently, it has been shown that HCMV pp65 can block alpha interferon-induced expression of the MxA gene and that a pp65-deficient virus has increased NF-{kappa}B DNA binding activity (12). However, pp65 overexpression did not downregulate TNFR1 surface expression in our hands (data not shown), and AD169var-T expresses pp65 with similar kinetics as AD169 (ATCC) (Fig. 5F). Furthermore, it has recently been shown by others that pp65 is unlikely to be a candidate for NF-{kappa}B regulation (56). Similarly, TNFR1 surface expression was not downregulated by IE2 after TNF-{alpha} induction in our hands (data not shown). Moreover, IE2, which has recently been reported to block beta interferon production (55), was expressed with similar kinetics in AD169 (ATCC)- and AD169var-T-infected cells (Fig. 5F). Thus, pp65 and IE2 are unlikely candidates for the viral inhibitory effects on NF-{kappa}B activation, and these findings further suggest that HCMV has obviously evolved several independent ways to contribute to the inhibition of cellular activities that support the innate immune response. Together our results suggest that one or more viral gene products expressed with early kinetics are primarily responsible for counteracting TNF-{alpha}-induced NF-{kappa}B signaling. At first sight this differs from what Jarvis and coworkers reported recently; they found that in their experimental system a true late gene product is responsible for this effect (36). Currently, we cannot be certain how this difference can be explained, particularly given the consistency of results between these two studies as a whole. However, a clue might come from our observation that NF-{kappa}B signaling was not completely inhibited when infected cells were treated with GCV (Fig. 7C). This may point to additional gene products expressed with late kinetics that are required for the full inhibitory effect. Possibly, these true late gene products might be the prevailing force under certain (experimental) conditions, which may explain the differences in this respect between the report by Jarvis et al. and the data presented here. Consistent with an earlier report by Baillie et al. (6) we find that downregulation of TNFR1 is independent of GCV treatment. Therefore, both the inhibition of NF-{kappa}B signaling and TNFR1 downregulation occur with early kinetics, which further rationally links these two activities. Interestingly, inhibition of TNFR1 surface expression as a function of early gene products is complete (Fig. 7D), whereas NF-{kappa}B signaling under GCV treatment is not (see above). The most direct interpretation of these results and an appealing hypothesis to bring together the present data and the published work by Jarvis et al. and Baillie et al. is that an early gene product is responsible for inhibiting NF-{kappa}B signaling via downregulation of TNFR1 and that this gene product collaborates with an additional late gene product that antagonizes NF-{kappa}B signaling in a TNFR1-independent manner. The HCMV variant should prove helpful in the future to decipher the viral gene products involved in antagonizing TNF-{alpha}-mediated NF-{kappa}B activation.

Despite the fact that TNF-{alpha}, IL-1ß, and LPS all signal through the classical NF-{kappa}B pathway and, therefore, have many factors in common that could be targeted by HCMV, the virus appears to use different mechanisms that block signal transduction upstream of the convergence point, i.e., the IKK complex. The most compelling evidence for this again comes from AD169var-T, which has lost the ability to downregulate surface expression of TNFR1 (Fig. 6). This lack of inhibition correlates well with sustained NF-{kappa}B activity after TNF-{alpha} stimulation in cells infected with AD169var-T. It strongly suggests that the lack of TNFR1 downregulation and sustained NF-{kappa}B activation are causally connected. Although TNFR1 downregulation in HCMV-infected cells has previously been shown, neither the responsible viral proteins nor the effect on NF-{kappa}B signaling was examined in that study (6). However, it was shown that TNFR1 downregulation was functionally relevant since the Jun N-terminal kinase, which is normally induced by TNFR1 signaling (16), was greatly inhibited by HCMV in TNF-{alpha}-stimulated cells (6). Thus, the HCMV variant rationally links the viral block to NF-{kappa}B activation after TNF-{alpha} stimulation to the downregulation of TNFR1 surface expression by HCMV.

The finding that AD169var-T is a loss-of-function variant defective in antagonizing NF-{kappa}B activation after TNF-{alpha} induction but not after IL-1ß induction also indicates that the block in IL-1ß- and LPS-mediated NF-{kappa}B activation is located upstream of the convergence point of the pathway, i.e., the IKK complex. As outlined above IL-1ß signaling and TLR signaling rely on the same set of adaptor proteins, and each of these could in principle be targeted by HCMV. Candidates include MyD88, a protein directly interacting with the cytoplasmic domain of TLR/IL-1R, which is crucially required for IL-1ß signaling to NF-{kappa}B (41), and IRAKs 1 and 4 (35), which recruit the ubiquitin E3 ligase TRAF 6 (13). How this complex links onto the IKK is not entirely clear but seems to involve the activation of the mitogen-activated protein kinase kinase kinase TAK1 (17, 54). In light of the downregulation of TNFR1 it would also be conceivable that HCMV inhibits the (surface) expression of the TLR/IL-1R. However, so far we have been unable to detect significant signals of IL-1R even on noninfected cells due to the limitation of appropriate reagents.

Disregarding the particular mechanisms involved, the HCMV-mediated block in NF-{kappa}B activation following proinflammatory stimulation adds a further level of complexity to the regulatory network this virus imposes on its host's antiviral defense system (28, 39). This complexity is also reflected by several seemingly contradictory reports supporting either a positive (14, 21) or negative (or neutral) (8, 25, 33) role for NF-{kappa}B during HCMV infection. The finding that inhibition of NF-{kappa}B signaling after proinflammatory stimulation is a function of later viral gene expression may help to rationalize this conundrum in that NF-{kappa}B activity may be beneficial in the early or reactivating phase of infection but disadvantageous at later stages or during persistent infection.


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ACKNOWLEDGMENTS
 
We are grateful to Bodo Plachter, Hartmut Hengel, and the late Susanna Prösch for virus stocks and to Claus Scheidereit for the gift of GST-I{kappa}B{alpha} constructs.

The work was supported through a grant from the Deutsche Forschungsgemeinschaft to C.H. (SFB 421 "Protektive und Pathologische Folgen der Antigenverarbeitung" TP-A12).


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Molecular Biology, Children's Hospital, Charité-CCM, Ziegelstrasse 5-9, Humboldt University Berlin, D-10098 Berlin, Germany. Phone: 49 (0)30 450 566 041. Fax: 49 (0)30 450 566 913. E-mail: christian.hagemeier{at}charite.de. Back

{triangledown} Published ahead of print on 27 September 2006. Back


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REFERENCES
 
    1
  1. Abate, D. A., S. Watanabe, and E. S. Mocarski. 2004. Major human cytomegalovirus structural protein pp65 (ppUL83) prevents interferon response factor 3 activation in the interferon response. J. Virol. 78:10995-11006.[Abstract/Free Full Text]
    2. 2
  2. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami, K. Nakanishi, and S. Akira. 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9:143-150.[CrossRef][Medline]
    3. 3
  3. Aggarwal, B. B. 2003. Signalling pathways of the TNF superfamily: a double-edged sword. Nat. Rev. Immunol. 3:745-756.[CrossRef][Medline]
    4. 4
  4. Alcami, A. 2003. Viral mimicry of cytokines, chemokines and their receptors. Nat. Rev. Immunol. 3:36-50.[CrossRef][Medline]
    5. 5
  5. Amici, C., G. Belardo, A. Rossi, and M. G. Santoro. 2001. Activation of I{kappa}b kinase by herpes simplex virus type 1. A novel target for anti-herpetic therapy. J. Biol. Chem. 276:28759-28766.[Abstract/Free Full Text]
    6. 6
  6. Baillie, J., D. A. Sahlender, and J. H. Sinclair. 2003. Human cytomegalovirus infection inhibits tumor necrosis factor alpha (TNF-{alpha}) signaling by targeting the 55-kilodalton TNF-{alpha} receptor. J. Virol. 77:7007-7016.[Abstract/Free Full Text]
    7. 7
  7. Baud, V., and M. Karin. 2001. Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol. 11:372-377.[CrossRef][Medline]
    8. 8
  8. Benedict, C. A., A. Angulo, G. Patterson, S. Ha, H. Huang, M. Messerle, C. F. Ware, and P. Ghazal. 2004. Neutrality of the canonical NF-{kappa}B-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro. J. Virol. 78:741-750.[Abstract/Free Full Text]
    9. 9
  9. Beutler, B. 2000. Tlr4: central component of the sole mammalian LPS sensor. Curr. Opin. Immunol. 12:20-26.[CrossRef][Medline]
    10. 10
  10. Bonizzi, G., and M. Karin. 2004. The two NF-{kappa}B activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25:280-288.[CrossRef][Medline]
    11. 11
  11. Boyle, K. A., R. L. Pietropaolo, and T. Compton. 1999. Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol. Cell. Biol. 19:3607-3613.[Abstract/Free Full Text]
    12. 12
  12. Browne, E. P., B. Wing, D. Coleman, and T. Shenk. 2001. Altered cellular mRNA levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mRNAs. J. Virol. 75:12319-12330.[Abstract/Free Full Text]
    13. 13
  13. Cao, Z., J. Xiong, M. Takeuchi, T. Kurama, and D. V. Goeddel. 1996. TRAF6 is a signal transducer for interleukin-1. Nature 383:443-446.[CrossRef][Medline]
    14. 14
  14. Caposio, P., M. Dreano, G. Garotta, G. Gribaudo, and S. Landolfo. 2004. Human cytomegalovirus stimulates cellular IKK2 activity and requires the enzyme for productive replication. J. Virol. 78:3190-3195.[Abstract/Free Full Text]
    15. 15
  15. Challacombe, J. F., A. Rechtsteiner, R. Gottardo, L. M. Rocha, E. P. Browne, T. Shenk, M. R. Altherr, and T. S. Brettin. 2004. Evaluation of the host transcriptional response to human cytomegalovirus infection. Physiol. Genomics 18:51-62.[Abstract/Free Full Text]
    16. 16
  16. Chen, G., and D. V. Goeddel. 2002. TNF-R1 signaling: a beautiful pathway. Science 296:1634-1635.[Abstract/Free Full Text]
    17. 17
  17. Chen, Z. J. 2005. Ubiquitin signalling in the NF-{kappa}B pathway. Nat. Cell Biol. 7:758-765.[CrossRef][Medline]
    18. 18
  18. Compton, T. 2004. Receptors and immune sensors: the complex entry path of human cytomegalovirus. Trends Cell Biol. 14:5-8.[CrossRef][Medline]
    19. 19
  19. Compton, T., E. A. Kurt-Jones, K. W. Boehme, J. Belko, E. Latz, D. T. Golenbock, and R. W. Finberg. 2003. Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2. J. Virol. 77:4588-4596.[Abstract/Free Full Text]
    20. 20
  20. Dargan, D. J., F. E. Jamieson, J. MacLean, A. Dolan, C. Addison, and D. J. McGeoch. 1997. The published DNA sequence of human cytomegalovirus strain AD169 lacks 929 base pairs affecting genes UL42 and UL43. J. Virol. 71:9833-9836.[Abstract]
    21. 21
  21. DeMeritt, I. B., J. P. Podduturi, A. M. Tilley, M. T. Nogalski, and A. D. Yurochko. 2006. Proloned activation of NF-{kappa}B by human cytomegalovirus promotes efficient viral replication and late gene expression. Virology 346:15-31.[CrossRef][Medline]
    22. 22
  22. DeMeritt, I. B., L. E. Milford, and A. D. Yurochko. 2004. Activation of the NF-{kappa}B pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78:4498-4507.[Abstract/Free Full Text]
    23. 23
  23. Eickhoff, J., M. Hanke, M. Stein-Gerlach, T. P. Kiang, K. Herzberger, P. Habenberger, S. Muller, B. Klebl, M. Marschall, T. Stamminger, and M. Cotton. 2004. RICK activates a NF-{kappa}B-dependent anti-human cytomegalovirus response. J. Biol. Chem. 279:9642-9652.[Abstract/Free Full Text]
    24. 24
  24. Feire, A. L., H. Koss, and T. Compton. 2004. Cellular integrins function as entry receptors for human cytomegalovirus via a highly conserved disintegrin-like domain. Proc. Natl. Acad. Sci. USA 101:15470-15475.[Abstract/Free Full Text]
    25. 25
  25. Ghazal, P., M. Messerle, K. Osborn, and A. Angulo. 2003. An essential role of enhancer for murine cytomegalovirus in vivo growth and pathogenesis. J. Virol. 77:3217-3228.[Abstract/Free Full Text]
    26. 26
  26. Ghosh, S., M. J. May, and E. B. Kopp. 1998. NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225-260.[CrossRef][Medline]
    27. 27
  27. Grundy, J. E., K. M. Lawson, L. P. MacCormac, J. M. Fletcher, and K. L. Yong. 1998. Cytomegalovirus-infected endothelial cells recruit neutrophils by the secretion of C-X-C chemokines and transmit virus by direct neutrophil-endothelial cell contact and during neutrophil transendothelial migration. J. Infect. Dis. 177:1465-1474.[Medline]
    28. 28
  28. Gutermann, A., A. Bubeck, M. Wagner, U. Reusch, C. Menard, and U. H. Koszinowski. 2002. Strategies for the identification and analysis of viral immune-evasive genes—cytomegalovirus as an example. Curr. Top. Microbiol. Immunol. 269:1-22.[Medline]
    29. 29
  29. Hayden, M. S., and S. Ghosh. 2004. Signaling to NF-{kappa}B. Genes Dev. 18:2195-2224.[Abstract/Free Full Text]
    30. 30
  30. Hirsch, A. J., and T. Shenk. 1999. Human cytomegalovirus inhibits transcription of the CC chemokine MCP-1 gene. J. Virol. 73:404-410.[Abstract/Free Full Text]
    31. 31
  31. Hiscott, J., H. Kwon, and P. Genin. 2001. Hostile takeovers: viral appropriation of the NF-{kappa}B pathway. J. Clin. Investig. 107:143-151.[CrossRef][Medline]
    32. 32
  32. Hsu, H., H. B. Shu, M. G. Pan, and D. V. Goeddel. 1996. TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84:299-308.[CrossRef][Medline]
    33. 33
  33. Isomura, H., T. Tsurumi, and M. F. Stinski. 2004. Role of the proximal enhancer of the major immediate-early promoter in human cytomegalovirus replication. J. Virol. 78:12788-12799.[Abstract/Free Full Text]
    34. 34
  34. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20:197-216.[CrossRef][Medline]
    35. 35
  35. Janssens, S., and R. Beyaert. 2003. Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members. Mol. Cell 11:293-302.[CrossRef][Medline]
    36. 36
  36. Jarvis, M. A., J. A. Borton, A. M. Keech, J. Wong, W. J. Britt, B. E. Magun, and J. A. Nelson. 2006. Human cytomegalovirus attenuates interleukin-1ß and tumor necrosis factor alpha proinflammatory signaling by inhibition of NF-{kappa}B activation. J. Virol. 80:5588-5598.[Abstract/Free Full Text]
    37. 37
  37. Karin, M., and Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-[{kappa}]B activity. Annu. Rev. Immunol. 18:621-663.[CrossRef][Medline]
    38. 38
  38. Maniatis, T., J. V. Falvo, T. H. Kim, T. K. Kim, C. H. Lin, B. S. Parekh, and M. G. Wathelet. 1998. Structure and function of the interferon-ß enhanceosome. Cold Spring Harbor Symp. Quant. Biol. 63:609-620.[CrossRef][Medline]
    39. 39
  39. Mocarski, E. S., Jr. 2002. Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol. 10:332-339.[CrossRef][Medline]
    40. 40
  40. Mogensen, T. H., J. Melchjorsen, L. Malmgaard, A. Casola, and S. R. Paludan. 2004. Suppression of proinflammatory cytokine expression by herpes simplex virus type 1. J. Virol. 78:5883-5890.[Abstract/Free Full Text]
    41. 41
  41. Murphy, E., D. Yu, J. Grimwood, J. Schmutz, M. Dickson, M. A. Jarvis, G. Hahn, J. A. Nelson, R. M. Myers, and T. E. Shenk. 2003. Coding potential of laboratory and clinical strains of human cytomegalovirus. Proc. Natl. Acad. Sci. USA 100:14976-14981.[Abstract/Free Full Text]
    42. 42
  42. Nabel, G., and D. Baltimore. 1987. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature 326:711-713.[CrossRef][Medline]
    43. 43
  43. Pahl, H. L. 1999. Activators and target genes of Rel/NF-{kappa}B transcription factors. Oncogene 18:6853-6866.[CrossRef][Medline]
    44. 44
  44. Parry, C. M., J. P. Simas, V. P. Smith, C. A. Stewart, A. C. Minson, S. Efstathiou, and A. Alcami. 2000. A broad spectrum secreted chemokine binding protein encoded by a herpesvirus. J. Exp. Med. 191:573-578.[Abstract/Free Full Text]
    45. 45
  45. Prichard, M. N., M. E. Penfold, G. M. Duke, R. R. Spaete, and G. W. Kemble. 2001. A review of genetic differences between limited and extensively passaged human cytomegalovirus strains. Rev. Med. Virol. 11:191-200.[CrossRef][Medline]
    46. 46
  46. Prosch, S., K. Staak, J. Stein, C. Liebenthal, T. Stamminger, H. D. Volk, and D. H. Kruger. 1995. Stimulation of the human cytomegalovirus IE enhancer/promoter in HL-60 cells by TNF{alpha} is mediated via induction of NF-{kappa}B. Virology 208:197-206.[CrossRef][Medline]
    47. 47
  47. Prosch, S., R. Wuttke, D. H. Kruger, and H. D. Volk. 2002. NF-{kappa}B—a potential therapeutic target for inhibition of human cytomegalovirus (re)activation? Biol. Chem. 383:1601-1609.[CrossRef][Medline]
    48. 48
  48. Sambucetti, L. C., J. M. Cherrington, G. W. Wilkinson, and E. S. Mocarski. 1989. NF-{kappa}B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO J. 8:4251-4258.[Medline]
    49. 49
  49. Santoro, M. G., A. Rossi, and C. Amici. 2003. NF-{kappa}B and virus infection: who controls whom. EMBO J. 22:2552-2560.[CrossRef][Medline]
    50. 50
  50. Silverman, N., and T. Maniatis. 2001. NF-{kappa}B signaling pathways in mammalian and insect innate immunity. Genes Dev. 15:2321-2342.[Free Full Text]
    51. 51
  51. Simmen, K. A., J. Singh, B. G. Luukkonen, M. Lopper, A. Bittner, N. E. Miller, M. R. Jackson, T. Compton, and K. Fruh. 2001. Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B. Proc. Natl. Acad. Sci. USA 98:7140-7145.[Abstract/Free Full Text]
    52. 52
  52. Sugano, N., W. Chen, M. L. Roberts, and N. R. Cooper. 1997. Epstein-Barr virus binding to CD21 activates the initial viral promoter via NF-{kappa}B induction. J. Exp. Med. 186:731-737.[Abstract/Free Full Text]
    53. 53
  53. Tabeta, K., P. Georgel, E. Janssen, X. Du, K. Hoebe, K. Crozat, S. Mudd, L. Shamel, S. Sovath, J. Goode, L. Alexopoulou, R. A. Flavell, and B. Beutler. 2004. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc. Natl. Acad. Sci. USA 101:3516-3521.[Abstract/Free Full Text]
    54. 54
  54. Takaesu, G., S. Kishida, A. Hiyama, K. Yamaguchi, H. Shibuya, K. Irie, J. Ninomiya-Tsuji, and K. Matsumoto. 2000. TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway. Mol. Cell 5:649-658.[CrossRef][Medline]
    55. 55
  55. Taylor, R. T., and W. A. Bresnahan. 2005. Human cytomegalovirus immediate early 2 gene expression blocks virus-induced beta interferon production. J. Virol. 79:3873-3877.[Abstract/Free Full Text]
    56. 56
  56. Taylor, R. T., and W. A. Bresnahan. 2006. Human cytomegalovirus immediate-early 2 protein IE86 blocks virus-induced chemokine expression. J. Virol. 80:920-928.[Abstract/Free Full Text]
    57. 57
  57. Wang, D., W. Bresnahan, and T. Shenk. 2004. Human cytomegalovirus encodes a highly specific RANTES decoy receptor. Proc. Natl. Acad. Sci. USA 101:16642-16647.[Abstract/Free Full Text]
    58. 58
  58. Wang, X., D. Y. Huang, S. M. Huong, and E. S. Huang. 2005. Integrin {alpha}vß3 is a coreceptor for human cytomegalovirus. Nat. Med. 11:515-521.[CrossRef][Medline]
    59. 59
  59. Wang, X., S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. S. Huang. 2003. Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus. Nature 424:456-461.[CrossRef][Medline]
    60. 60
  60. Wiebusch, L., R. Uecker, and C. Hagemeier. 2003. Human cytomegalovirus prevents replication licensing by inhibiting MCM loading onto chromatin. EMBO Rep. 4:42-46.[CrossRef][Medline]
    61. 61
  61. Yurochko, A. D., E. S. Hwang, L. Rasmussen, S. Keay, L. Pereira, and E. S. Huang. 1997. The human cytomegalovirus UL55 (gB) and UL75 (gH) glycoprotein ligands initiate the rapid activation of Sp1 and NF-{kappa}B during infection. J. Virol. 71:5051-5059.[Abstract]
    62. 62
  62. Zhang, S. Q., A. Kovalenko, G. Cantarella, and D. Wallach. 2000. Recruitment of the IKK signalosome to the p55 TNF receptor: RIP and A20 bind to NEMO (IKK{gamma}) upon receptor stimulation. Immunity 12:301-311.[CrossRef][Medline]
    63. 63
  63. Zhu, H., J. P. Cong, G. Mamtora, T. Gingeras, and T. Shenk. 1998. Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95:14470-14475.[Abstract/Free Full Text]

Journal of Virology, December 2006, p. 11686-11698, Vol. 80, No. 23
0022-538X/06/$08.00+0     doi:10.1128/JVI.01168-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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