Hepatitis C Virus Core Protein Suppresses NF-B Act
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病菌学杂志 2005年第12期
Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University School of Medicine, and Department of Veterans Affairs Medical Center, Nashville, Tennessee 37232
Department of Microbiology and Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22908
ABSTRACT
In addition to hepatocytes, hepatitis C virus (HCV) infects immune cells, including macrophages. However, little is known concerning the impact of HCV infection on cellular functions of these immune effector cells. Lipopolysaccharide (LPS) activates IB kinase (IKK) signalsome and NF-B, which leads to the expression of cyclooxygenase-2 (COX-2), which catalyzes production of prostaglandins, potent effectors on inflammation and possibly hepatitis. Here, we examined whether expression of HCV core interferes with IKK signalsome activity and COX-2 expression in activated macrophages. In reporter assays, HCV core inhibited NF-B activation in RAW 264.7 and MH-S murine macrophage cell lines treated with bacterial LPS. HCV core inhibited IKK signalsome and IKK? kinase activities induced by tumor necrosis factor alpha in HeLa cells and coexpressed IKK in 293 cells, respectively. HCV core was coprecipitated with IKK? and prevented nuclear translocation of IKK?. NF-B activation by either LPS or overexpression of IKK? was sufficient to induce robust expression of COX-2, which was markedly suppressed by ectopic expression of HCV core. Together, these data indicate that HCV core suppresses IKK signalsome activity, which blunts COX-2 expression in macrophages. Additional studies are necessary to determine whether interrupted COX-2 expression by HCV core contributes to HCV pathogenesis.
INTRODUCTION
Hepatitis C virus (HCV), a flavivirus, causes hepatitis, cirrhosis, and hepatocellular carcinoma (18). Currently, almost 3% of the world population is infected by HCV, and these numbers seem to be increasing (3). One of the most remarkable features of HCV infection is that more than 85% of acutely infected patients become chronically infected (4). Although CD4+ and CD8+ T-cell responses are crucial for controlling HCV infection in acute HCV patients, these T-cell responses are significantly impaired in chronic HCV patients (16). Thus, this suggests that HCV evades host immune responses. While hepatocytes are a major target of HCV infection, recent studies showed that HCV can replicate in immune cells such as B and T lymphocytes and monocytes that express HCV receptors, such as CD81 and low-density lipoprotein receptor (1, 2, 52). Thus, it is possible that HCV infects immune effector cells, which contributes to evasion of host immune surveillance.
The HCV core protein, a nucleocapsid protein, binds to the cytoplasmic domain of tumor necrosis factor receptor (TNFR) and lymphotoxin-beta receptor to regulate apoptosis (16, 17, 45, 81). This viral protein is also involved in oncogenesis, as evidenced by the development of hepatocellular carcinoma in transgenic mice expressing HCV core in the liver (47). In addition, HCV core has been shown to affect diverse cellular and viral gene expressions (53, 55, 60) and, depending on subtypes, to activate or suppress NF-B function that is involved in both innate and adaptive immunity (27, 44, 61, 79).
NF-B is a homo- or heterodimer of five proteins: c-Rel, RelA (p65), RelB, p50, and p52. Under unstimulated conditions, NF-B resides in the cytoplasm by forming complexes with inhibitory B (IB) (27). Proinflammatory stimuli, such as TNF- and lipopolysaccharide (LPS), induce signal cascades through their cognate receptors, TNFR and Toll-like receptor 4 (TLR4), to activate IB kinase (IKK) signalsome and subsequently NF-B that is required for innate and adaptive immune responses to pathogens (13, 38). IKK signalsome is composed of at least two kinases, IKK and -?, and a regulatory factor, IKK (26, 36). Among these proteins, IKK? is known as a major kinase and IKK is required for the full activation of IKK? upon proinflammatory stimulation (41-43, 56, 57, 59, 66, 78). Treatment of cells with TNF- results in IKK signalsome recruitment to the type 1 TNF- receptor (TNFR1), which induces phosphorylation at key residues in the activation loop of the kinase domain of IKK? to be an active kinase (21). Activated IKK signalsome phosphorylates IB, which subsequently undergoes ubiquitination and degradation, liberating NF-B to translocate into the nucleus and activate target genes (37).
NF-B influences gene expression of cyclooxygenase-2 (COX-2) along with other transcription factors, including CREB and C/EBP-? (15, 20, 33, 34, 48, 50, 63, 67, 72, 75). Accordingly, proinflammatory stimuli induce the expression of COX-2 to catalyze the production of prostaglandins, which promote inflammation through a variety of mechanisms.
Our hypothesis is that HCV evades innate immunity by directly infecting immune cells and altering gene expression of key inflammatory molecules. Here we tested whether the HCV core protein affects NF-B activity and COX-2 production in macrophages. We demonstrate that the HCV core protein interacts with IKK?, suppresses its kinase activity, and interferes with nuclear translocation of IKK?, which are correlated with inhibition of NF-B activity. Furthermore, we show that the HCV core protein suppresses NF-B-dependent COX-2 expression. These data indicate that the HCV core protein interrupts diverse IKK? functions, which results in blunted COX-2 expression.
MATERIALS AND METHODS
Cell lines. Murine macrophage cell line RAW 264.7, murine alveolar macrophage cell line MH-S, and HEK 293 and HeLa cells were all obtained from the American Type Culture Collection (Rockville, MD). Cell lines were maintained according to the recommendations of the American Type Culture Collection.
Plasmids. The coding sequence of HCV core (Hutchinson strain, genotype 1a) was inserted into the polylinker site of pCI:neo (Promega). The resultant construct, named pCI-HCV core, was engineered to express the 192-amino-acid-long HCV core protein. To generate a construct that encodes FLAG-tagged HCV core, pCI-HCV core was digested with restriction enzymes NheI and EcoRI (New England BioLabs) and inserted with a linker containing the FLAG sequence. The coding sequence of HCV core and E1 (Hutchinson strain, genotype 1a) was amplified by Pfu polymerase (Stratagene). The forward primer binds to the first 20 nucleotides of the 5' end of the HCV core sequence and contains NheI restriction site, Kozak, and FLAG sequences (5'-GCGGCTAGCCACCATGGACTACAAAGACGATGACGATAAAGGGATGAGCACGAATCCTAAACC-3'; underlined is the core sequence). The reverse primer binds to 18 nucleotides of the 3' end of the HCV E1 sequence and contains HindIII (5'-GCAAGCTTGCCGGCAAATAGCAGCAG-3'). The PCR product was digested with restriction enzymes NheI and HindIII (New England BioLabs) and inserted into the corresponding sites of pcDNA3(-)-Myc/His (Invitrogen). Candidate clones encoding HCV core and E1 were analyzed by sequencing (Vanderbilt DNA Sequencing Core Facility). Expression of the FLAG-tagged HCV core and Myc-tagged protein was determined by Western blotting with M2 antibody (Sigma) and anti-Myc antibody (Santa Cruz Biotechnology). Plasmids encoding a FLAG-tagged IKK and IKK? were kindly provided by R. Carter (Vanderbilt University) and F. Mercurio (Signal Research Division, Celgene Corp., New Jersey), respectively, and a plasmid expressing T7-tagged NEMO/IKK was a gift from E. S. Alnemri (Thomas Jefferson University). A plasmid encoding superrepressor IBS32,36A was a gift from K. Pennington (Vanderbilt University). The luciferase reporter plasmid NF-B-Luc was purchased from Stratagene (La Jolla, CA).
Plasmid transfection, LPS treatment, and luciferase assay. Cells were transfected with Superfect (QIAGEN), as specified by the manufacturer. Each transfection was normalized with appropriate empty vector plasmids. At 48 h posttransfection, transfected cells were harvested for the experiment. For the LPS treatment, after incubation for 14 to 16 h in a 37°C, CO2 incubator, transfected cells were further maintained without serum overnight and subsequently treated with LPS (1 μg/ml in phosphate-buffered saline; Sigma) for 4 h to induce COX-2 expression. For the luciferase assay, cells were cotransfected with luciferase reporter plasmids NF-B-Luc and tk-Renilla Luc. Luciferase activity was measured by a dual-luciferase assay system (Promega) according to the protocol recommended by the manufacturer. NF-B-mediated luciferase activity was normalized with Renilla luciferase activity. The luciferase assay was done in triplicate.
Viruses and infection. Adenoviruses encoding IKK?, superrepressor IkBS32,36A, and ?-galactosidase (?-Gal) were described previously (58). Viruses were propagated and virus titer was determined by standard protocols. RAW 264.7 cells were infected with adenoviruses at multiplicity of infection of 0.2 for 1 h. After inocula were removed, cells were placed in fresh medium and incubated overnight at 37°C in a CO2 incubator. At 24 h postinfection, cells were harvested for the experiment.
Immunoprecipitation and Western blotting. Total cell lysate was prepared with RIPA (radioimmunoprecipitation assay) cell lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1% sodium orthovanadate, 1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) supplemented with phenylmethylsulfonyl fluoride (1 mM), aprotonin (2 μg/ml), leupeptin (2 μg/ml), and soybean trypsin inhibitor (37.5 μg/ml). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Specific bands were revealed using enhanced chemiluminescence (ECL plus; Amersham). For immunoprecipitation analysis, 1 to 2 μg of appropriate antibodies was added to precleared cell lysate that was normalized by protein content and incubated overnight at 4°C. Immune complexes were captured with 30 μl of protein A-Sepharose (Zymed) for 30 min at 4°C and washed five times with RIPA buffer. Antibodies used in this study were as follows: polyclonal anti-murine COX-2 (Cayman Chemical); monoclonal anti-FLAG M2 antibody (Sigma); polyclonal anti-IKK/? and anti-IKK (Santa Cruz Biotechnology); monoclonal anti-IKK and -? (PharMingen); monoclonal anti-HCV core antibody (Affinity Bioreagents, Inc.).
IKK assay. Transfected HeLa cells were treated with 10 ng/ml of TNF- (R&D Systems Inc.) for 15 min before lysis. Cytoplasmic extracts from HeLa or HEK 293 cells were prepared as described previously (49). To immunoprecipitate IKK signalsome, ELB buffer (49) was added to the prepared cytoplasmic extract. Kinase activity was measured in a reaction buffer containing 10 mM HEPES (pH 7.4), 5 mM MgCl2, 1 mM MnCl2, ATP (10 μM), [-32P]ATP (5 μCi), and recombinant glutathione S-transferase (GST)-IB, GST protein fused to amino acids 1 to 54 of IB, at 30°C for 30 min. Radiolabeled products were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and revealed by autoradiography.
Preparation of cytoplasmic and nuclear fractions. Cytoplasmic and nuclear fractions were prepared as described elsewhere (9). In brief, cells were washed three times with ice-cold phosphate-buffered saline and added to hypotonic buffer (10 mM HEPES [pH 7.9], 5 mM KCl, 1.5 mM MgCl2, 1 mM NaF, and 1 mM Na3VO4) supplemented with 1 mM dithiothreitol, phenylmethylsulfonyl fluoride (1 mM), aprotonin (2 μg/ml), leupeptin (2 μg/ml), and soybean trypsin inhibitor (37.5 μg/ml). After lysis for 15 min on ice, the cytoplasmic fraction was prepared by centrifugation at 3,000 rpm for 5 min. The cytoplasmic fraction was cleared by centrifugation at 13,000 rpm for 15 min before further analysis. To prepare the nuclear fraction, the cell pellet generated after initial centrifugation was washed with the hypotonic buffer three times and suspended in high-salt buffer (20 mM HEPES [pH 7.9], 1.5 mM MgCl2, 0.2 mM EDTA [pH 8.0], 420 mM NaCl, 25% glycerol [vol/vol], 50 mM ?-glycerophosphate, 1 mM NaF, and 1 mM Na3VO4) supplemented with 1 mM dithiothreitol, phenylmethylsulfonyl fluoride (1 mM), aprotonin (2 μg/ml), leupeptin (2 μg/ml), and soybean trypsin inhibitor (37.5 μg/ml). The suspended cell pellet was incubated for 30 min on ice with occasional vortexing, and the nuclear fraction was collected after centrifugation at 13,000 rpm for 10 min. Protein content from each fraction was quantified by the Bradford assay (Bio-Rad) as specified by the manufacturer to ensure equal loading.
Statistical analysis. For comparison among groups, paired or unpaired t tests and one-way analysis of variance tests were used (with the assistance of InStat; Graphpad Software, Inc., San Diego, CA). P values of <0.05 are considered significant.
RESULTS
HCV core suppresses NF-B activity in LPS-treated macrophages. HCV core differentially modulates NF-B activity, depending on the subtype of HCV (54). To examine whether the HCV core used in this study (genotype 1a) interferes with NF-B activity in LPS-treated macrophages, RAW 264.7, a murine macrophage cell line, and MH-S, a murine alveolar macrophage cell line, were transfected with a plasmid encoding HCV core along with the NF-B-luciferase reporter construct in the presence or absence of LPS. The results in Fig. 1 showed that HCV core, by itself, did not activate NF-B but rather suppressed NF-B activity elicited by LPS treatment of RAW 264.7 (Fig. 1A) and MH-S (Fig. 1B) cells. A similar experiment using TNF- produced similar results (data not shown). These results demonstrate that HCV core suppresses NF-B activity in macrophages.
HCV core inhibits IKK signalsome kinase activity. To investigate the mechanism by which HCV core inhibits NF-B activity, we tested whether HCV core interferes with IKK kinase activity. For these studies, we employed HeLa and 293 cells because of the ability to achieve high transfection efficiencies in these cell lines. First, to test whether these cell lines were able to recapitulate the results shown in Fig. 1, we performed a similar experiment. As shown in Fig. 2A, when HeLa cells were treated with TNF-, NF-B-mediated transcriptional activity was increased (lanes 1 and 2). However, the presence of HCV core suppressed NF-B activity elicited by TNF- (lanes 2 and 4). Similarly, HCV core suppressed NF-B activated by TNF- in HEK 293 cells (Fig. 2B, lanes 2 and 3). These results suggest that HCV core also represses NF-B activity in these cell lines.
We tested whether coexpression of HCV core and E1 alters the effect of HCV core on NF-B activity. HeLa cells were transfected with a plasmid encoding HCV core and E1 along with the NF-B reporter construct and treated with TNF-. As shown in Fig. 2C, HCV core was able to suppress NF-B activity elicited by TNF- in the presence of HCV E1 (lane 4). A similar result was obtained in HEK 293 cells (data not shown). These results indicate that suppression of NF-B activity by HCV core is not significantly affected by E1.
Next, we tested whether HCV core interferes with IKK signalsome kinase activity. HeLa cells were transfected with increasing amounts of a plasmid encoding HCV core and treated with TNF- (10 ng/ml) for 15 min to induce IKK signalsome activity. The IKK signalsome was immunoprecipitated with anti-IKK? antibody, extensively washed, and incubated with GST-IB and [-32P]ATP for kinase assays. As shown in Fig. 3A, HCV core reduced IKK kinase activity elicited by TNF- treatment in a dose-dependent fashion.
Since IKK? is known as a major kinase in inflammatory gene expression and overexpression of IKK? results in kinase activity that is markedly increased by IKK (24), we examined whether HCV core inhibits IKK? kinase activity in the absence of an activating stimulus (Fig. 3B). HEK 293 cells were transfected with plasmids expressing FLAG-tagged IKK? and T7-tagged IKK in the presence or absence of HCV core. For IKK? kinase assays, IKK? was precipitated with M2 antibody. As shown in Fig. 3B, IKK? kinase activity was markedly enhanced by IKK (lane 2 compared to lane 1); however, this was strongly suppressed by HCV core (lane 3). Western blot analysis also revealed that the phosphorylated form of IKK?, an indicator of kinase activation, was induced by overexpression of IKK. The phosphorylation of IKK? was blocked by ectopic expression of the HCV core protein (middle panel, lanes 2 and 3). These results indicate that HCV core specifically suppresses IKK? kinase activity.
HCV core physically interacts with IKK?. To understand how the HCV core protein suppresses IKK? kinase activity, we examined the physical interaction between the two proteins by immunoprecipitation assay. HeLa cells were transfected with either an empty vector or a plasmid encoding FLAG-tagged HCV core. After cell lysis, the cytoplasmic fraction was incubated with M2 antibody to capture FLAG-tagged HCV core, and the immune complex was analyzed by SDS-PAGE and Western blotting. First, to detect any subunits of IKK signalsome interacting with the HCV core protein, the membrane was incubated with a mixture of anti-IKK, -IKK?, and -IKK antibodies. As shown in Fig. 4A, the HCV core protein coprecipitated with IKK and/or IKK? but not with IKK. To identify the precipitated band, coimmunoprecipitates of the HCV core protein were further analyzed by Western blotting with IKK and IKK? antibodies. The result in Fig. 4B indicated that IKK? but not IKK coimmunoprecipitated with the HCV core protein.
The HCV core interferes with nuclear localization of IKK?. A recent discovery that IKK and -? are located not only in the cytoplasm but also in the nucleus suggests additional functions of IKK signalsome in regulation of gene expression (12). Indeed, IKK is recruited to the NF-B-binding DNA element via physical interaction with CREB-binding protein, which enhances target gene expression (7, 76). IKK is also directly involved in transcription regulation by inhibiting the interaction between IKK and CREB-binding protein (71). Therefore, we investigated the possibility that the HCV core protein affects IKK? activity by interfering with nuclear-cytoplasmic partitioning of IKK?.
To test whether HCV core selectively interferes with IKK? partitioning within a cell, HEK 293 cells were transfected with plasmids encoding FLAG-tagged IKK and IKK? along with the plasmid encoding HCV core. To determine partitioning of IKK and IKK?, cytoplasmic and nuclear fractions were prepared and analyzed by Western blotting with M2 antibody to reveal IKK and -?. As shown in Fig. 5A, while the majority of IKK resides in the cytoplasm, IKK? was detected in both cytoplasmic and nuclear fractions (lanes 1, 2, 5, and 6). However, the presence of the HCV core protein decreased localization of IKK? in the nuclear fraction (lanes 6 and 8), while not significantly affecting the amount of cytoplasmic IKK?. To ensure equal loading of nuclear proteins, the membrane was stripped and probed with anti-YY1 antibody (8, 29, 46).
To further confirm this observation, a similar experiment was conducted to determine the distribution of endogenous IKK? (Fig. 5B). HEK 293 cells were transfected with increasing amounts of the HCV core-encoding plasmid. Transfected cells were treated with 10 μg/ml of TNF- to examine a possible effect on distribution of endogenous IKK?. Western blotting analysis in Fig. 5B showed that distribution of IKK? was not significantly affected by TNF- treatment (compare lanes 1 and 2 and lanes 5 and 6), as indicated in another study (7). However, the HCV core protein reduced nuclear localization of IKK? (lanes 6, 7, and 8). To ensure equal loading of nuclear protein, the membrane was treated as for Fig. 5A to reveal nuclear protein YY1. Taken together, these results indicate that the HCV core protein blocks IKK? shuttling to the nucleus.
NF-B activation elicited by IKK? is sufficient to induce COX-2. Involvement of NF-B in COX-2 expression is well documented in a variety of cell types (15, 20, 77).
To define the relationship between NF-B activation and COX-2 expression in RAW 264.7 cells, we treated the cells with endotoxin and measured COX-2 expression and NF-B activation by Western blot analysis. As indicated in Fig. 6A, COX-2 protein production increased in response to treatment with LPS, and this was associated with degradation of cytoplasmic IB and the appearance of immunoreactive RelA in the nucleus (Fig. 6B). These data show that the macrophage cell line treated with LPS results in both activation of NF-B and COX-2 protein production.
To assess the role of NF-B in COX-2 expression in macrophages without ambiguity associated with cross talk by inflammatory signals, NF-B was activated in a highly specific manner through ectopic expression of IKK? (26), and resultant COX-2 expression was analyzed by Western blotting in two separate murine macrophage cell lines, RAW 264.7 and MH-S. As shown in Fig. 7, ectopic expression of IKK? increases COX-2 protein production in both macrophage cell lines in a dose-dependent fashion. These data indicate that NF-B activation by ectopic expression of IKK? is sufficient to induce COX-2 expression.
To assess the specificity of this response, we examined whether superrepressor IkBS32,36A suppresses COX-2 expression induced by IKK?. First, RAW 264.7 cells were transfected with an NF-B luciferase reporter construct along with the IKK?-expressing plasmid in the absence or presence of the superrepressor-expressing plasmid. As shown in Fig. 8A, transfection of the superrepressor IBS32,36A markedly inhibits NF-B-mediated luciferase activity induced by IKK?. Next, to test whether COX-2 protein production induced by IKK? is suppressed by IBS32,36A, RAW 264.7 cells were infected with replication-deficient adenoviruses encoding kinase-active IKK?, IBS32,36A, or ?-Gal. Western blot analysis in Fig. 8B shows that infection with IKK?-expressing adenovirus induced COX-2 expression, while infection with ?-Gal-expressing adenovirus failed to do so. Coinfection with two adenoviruses encoding IKK? and IBS32,36A blunted the production of COX-2. Taken together, these data demonstrate that selective NF-B activation results in COX-2 expression in macrophage cell lines.
HCV core suppresses COX-2 expression induced by LPS. Finally, we examined whether the inhibitory effect of HCV core on NF-B activity leads to down regulation of COX-2 expression. RAW 264.7 cells were transfected with either an empty host plasmid or a plasmid expressing the HCV core protein. Subsequently, transfected cells were treated with LPS to induce COX-2. As indicated in Fig. 9, HCV core protein, by itself, did not induce COX-2 expression (lanes 3 and 4). In contrast, HCV core protein strongly suppressed COX-2 expression induced by LPS treatment (lanes 5 and 6).
DISCUSSION
Primary HCV infection evades protective immunity and establishes a chronic infection (4, 16). Chronic HCV-infected patients have reduced Th1-type CD4+ T-cell activity and increased Th2-type CD4+ T-cell activity (16, 23). Some chronic hepatitis C patients treated with alpha interferon (IFN-) (14) and ribavirin show enhanced Th1 CD4+ T-cell effector function with improvement of the medical condition (32, 65, 69). An HCV murine model also showed the compromised production of Th1 cytokines, such as interleukin-2 (IL-2) and IFN-, by HCV core (39). Similarly, there is a decreased Th1 cytokine production in transgenic mice that express HCV core (62). These data, together, suggest that HCV evades the host immune response through a mechanism that is mediated by HCV core. Recent experimental evidence shows that HCV infection occurs with a broad cellular tropism encompassing not only hepatocyte cells but also immune cells, such as B and T lymphocytes and monocytes (1). However, the impact of HCV on infected immune cells is not understood. In this study, we addressed this issue by examining an inhibitory effect of HCV core on NF-B, a key transcription factor in innate and adaptive immunity. Our data showed that, through inhibition of IKK kinase activity, HCV core hinders NF-B activity, which results in blunted expression of an important inflammatory protein, COX-2, in macrophages.
Along with constitutively expressed COX-1, COX-2 metabolizes arachidonic acid to prostaglandin H2, which is further metabolized to generate various prostanoids (24). Prostanoids comprise three groups: prostaglandins, such as D2 (PGD2), E2 (PGE2), and F2 (PGF2); prostacylin, such as PGI2; and thromboxane A2. In general, prostanoids are considered to promote inflammation by inducing vasodilation and increasing vascular permeability and cellular migration to the site of inflammation (22, 73, 74). Notably, prostanoids are also important modulators of adaptive immunity (28, 30); however, effects of prostanoids on immunity are complex, and the net result depends on the surrounding milieu. For example, PGE2 promotes dendritic cell maturation and migration to lymphoid organs in peripheral tissue, while it suppresses T-cell activation by dendritic cells in lymphoid organs (28). In the case of PGD2, while enhancing the Th2-type immune response, PGD2 and its derivative cyclopentenone prostaglandins are known to have anti-inflammatory activities by inhibiting NF-B, inducing apoptosis of macrophages and neutrophils in inflammatory lesions, and activating anti-inflammatory transcription factor PPAR- (64). Other prostanoids, such as PGI2 and thromboxane A2, also increase T-cell functions, and PGI2 enhances natural killer (NK) cell functions (19, 35, 68, 70). Nevertheless, these results clearly illustrate that prostanoids are implicated in innate and adaptive immunity.
It is not well documented that there is a relationship between HCV infection and COX-2 expression that might influence HCV pathogenesis. As yet, combined treatment with IFN- and ribavirin is the popular measure to control hepatitis C, albeit with limited success. A report that IFN- increased PGE2 production suggested that elevated PGE2 might be attributed to unresponsiveness to IFN- treatment, given the immunosuppressive nature of PGE2 (6). Accordingly, IFN- signaling can be enhanced by indomethacin, one of the nonsteroidal anti-inflammatory drugs (NSAIDs) that blocks prostanoids production by inhibiting COX-1 and COX-2 activities (25). However, combined treatment of HCV patients with NSAIDs and IFN- has shown limited success and is less effective than treatment with ribavirin (5). This result might be due to complex effects of prostanoids on host immunity. Interestingly, recent studies have shown that arachidonic acid is able to suppress HCV genomic replication and has a beneficial effect on HCV-infected patients (40, 51). Although it is not understood how arachidonic acid affects HCV, it is conceivable that impaired arachidonic acid metabolism might be favorable for HCV survival.
Our data implicate HCV in COX-2 expression. Although additional studies are apparently required to understand the biological significance of deregulated COX-2 expression in HCV-infected cells, possible roles of COX-2 in HCV pathogenesis can be hypothesized. It is possible that HCV avoids unwanted stimulation of adaptive immune responses against itself by down regulating COX-2 expression in HCV-infected antigen-presenting cells, such as macrophages and dendritic cells. Another possibility is that, by down regulating COX-2 expression, HCV-infected immune effector cells avoid lysis by NK cells and/or apoptosis caused by cyclopentenone 15d-PGJ2, which is derived from PGD2 during inflammatory responses. Lastly, HCV avoids evoking antiviral activities of prostanoids by suppressing COX-2 expression. Although a precise mechanism is not known, PGA and PGJ series prostaglandins have been reported to have antiviral activities against various DNA and RNA viruses (64). Recently, PGI2 was reported to have antiviral activity against respiratory syncytial virus in a respiratory syncytial virus-induced disease mouse model (31). Similarly, in a vaccinia virus-induced disease mouse model, PGI2-treated mice showed enhanced survival, whereas NSAIDs-treated mice succumbed to virus-induced mortality (80). Therefore, impaired COX-2 expression could be attributable to host immune evasion by HCV, favoring persistent HCV infection.
COX-2 expression is regulated by diverse transcription factors that include NF-B, CREB, and C/EBP-?. However, the relative importance of these pathways is not clear and, more importantly, the impact of NF-B on COX-2 expression in macrophages is controversial. Our results show that COX-2 is induced by selective activation of NF-B alone and is suppressed by expression of superrepressor IB, suggesting that NF-B activation is sufficient for COX-2 expression.
Our data in Fig. 8 show that COX-2 induction by LPS is substantially inhibited by HCV core. Given the complexity of cox-2 transcriptional regulation and the diverse transcriptional modulating function of HCV core, the possibility that HCV core also influences function of other transcription factors that are involved in COX-2 expression cannot be excluded. It is also possible that the significant decrease of COX-2 is due to a direct interruption of TNF- or other cytokine production, via NF-B suppression by HCV core, which otherwise contributes to COX-2 expression in an autocrine fashion, as LPS treatment induces TNF- and other cytokines that, along with TLR4 signaling, enhance COX-2 expression (11). Finally, it is conceivable that HCV core could alter the half-life or increase the turnover rate of COX-2 protein, although transcription of the cox-2 gene is closely related to the level of COX-2 protein expression (M. D. Bryer, personal communication), and the half-life of COX-2 mRNA is significantly prolonged by LPS treatment (10).
NF-B is either activated or suppressed by HCV core, depending on subtype. For example, while the core protein of HCV type 1b activates NF-B activity, that of HCV type 1a (Hutchinson strain) suppresses NF-B activity (54). A mutagenesis study showed that a single amino acid substitution in the core protein of HCV type 1a converting either from Lys at 9 (K9) to Arg or from Asn at 11 (N11) to Thr is sufficient to relieve its inhibitory function (54). However, it is not understood how these amino acid residues affect modulation of NF-B activity.
Previously, it was proposed that activation of NF-B by type 1b HCV core is related to IKK?, although a direct biochemical interaction between the two proteins and direct functional interference by the HCV core protein were not presented (79). In the present study, we report evidence that type 1a HCV core protein biochemically interacts with IKK? and inhibits IKK kinase activity (Fig. 6A and 7). Although we do not fully understand the mechanism that, unlike type 1a HCV core, type 1b HCV core activates IKK signalsome and NF-B activity, our data raise the possibility that a capability of HCV core to physically interact with IKK? might determine differential regulation of NF-B activity. It is possible that the type 1a HCV core protein sequesters IKK? and prevents formation of a fully functional IKK signalsome, which impairs NF-B signaling. Alternatively, it is possible that the HCV core protein interferes with stoichiometric interactions between IKK? and, subsequently, other subunits of IKK signalsome. More importantly, given that a single amino acid change at either K9 or N11 relieves inhibitory functions of the HCV core protein type 1a, it is conceivable that microenvironmental changes such as electric charge or physical hindrance affect interactions between IKK? and its substrates.
Another observation in our study that might lead to understanding of the mechanism by which HCV core suppresses IKK activity is the disruption of cytoplasmic-nuclear partitioning of IKK?. As shown in Fig. 4, either transfected or endogenous IKK-? is located in both cytoplasmic and nuclear fractions; however, the HCV core protein selectively disturbs the nuclear localization of IKK-?. In similar experiments to measure cellular distributions of IKK and IKK, HCV core did not affect the distributions of these two subunits of the IKK signalsome (data not shown). The functional significance of nuclear IKK? and the biological significance of blocked translocation of IKK? by the HCV core protein are areas of intense investigation.
In summary, we studied the inhibitory function of HCV core on NF-B activity in macrophages. Our data indicate that inhibition of NF-B activity by the HCV core protein might be related to its physical interaction with and interrupted nuclear localization of IKK?. Furthermore, our data show that COX-2 expression is induced by NF-B activation and suppressed by the HCV core protein. These results suggest the possibility that HCV-infected macrophages have impaired immune responses at least in part due to impaired COX-2 expression.
ACKNOWLEDGMENTS
This work was supported by the Department of Veterans Affairs and National Institutes of Health grants AI057591 (Y.S.H.), HL075557 (J.W.C.), and HL061419 (T.S.B.) and program project HL066196.
We thank Tamara Lasakow for editorial assistance with the manuscript.
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Department of Microbiology and Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22908
ABSTRACT
In addition to hepatocytes, hepatitis C virus (HCV) infects immune cells, including macrophages. However, little is known concerning the impact of HCV infection on cellular functions of these immune effector cells. Lipopolysaccharide (LPS) activates IB kinase (IKK) signalsome and NF-B, which leads to the expression of cyclooxygenase-2 (COX-2), which catalyzes production of prostaglandins, potent effectors on inflammation and possibly hepatitis. Here, we examined whether expression of HCV core interferes with IKK signalsome activity and COX-2 expression in activated macrophages. In reporter assays, HCV core inhibited NF-B activation in RAW 264.7 and MH-S murine macrophage cell lines treated with bacterial LPS. HCV core inhibited IKK signalsome and IKK? kinase activities induced by tumor necrosis factor alpha in HeLa cells and coexpressed IKK in 293 cells, respectively. HCV core was coprecipitated with IKK? and prevented nuclear translocation of IKK?. NF-B activation by either LPS or overexpression of IKK? was sufficient to induce robust expression of COX-2, which was markedly suppressed by ectopic expression of HCV core. Together, these data indicate that HCV core suppresses IKK signalsome activity, which blunts COX-2 expression in macrophages. Additional studies are necessary to determine whether interrupted COX-2 expression by HCV core contributes to HCV pathogenesis.
INTRODUCTION
Hepatitis C virus (HCV), a flavivirus, causes hepatitis, cirrhosis, and hepatocellular carcinoma (18). Currently, almost 3% of the world population is infected by HCV, and these numbers seem to be increasing (3). One of the most remarkable features of HCV infection is that more than 85% of acutely infected patients become chronically infected (4). Although CD4+ and CD8+ T-cell responses are crucial for controlling HCV infection in acute HCV patients, these T-cell responses are significantly impaired in chronic HCV patients (16). Thus, this suggests that HCV evades host immune responses. While hepatocytes are a major target of HCV infection, recent studies showed that HCV can replicate in immune cells such as B and T lymphocytes and monocytes that express HCV receptors, such as CD81 and low-density lipoprotein receptor (1, 2, 52). Thus, it is possible that HCV infects immune effector cells, which contributes to evasion of host immune surveillance.
The HCV core protein, a nucleocapsid protein, binds to the cytoplasmic domain of tumor necrosis factor receptor (TNFR) and lymphotoxin-beta receptor to regulate apoptosis (16, 17, 45, 81). This viral protein is also involved in oncogenesis, as evidenced by the development of hepatocellular carcinoma in transgenic mice expressing HCV core in the liver (47). In addition, HCV core has been shown to affect diverse cellular and viral gene expressions (53, 55, 60) and, depending on subtypes, to activate or suppress NF-B function that is involved in both innate and adaptive immunity (27, 44, 61, 79).
NF-B is a homo- or heterodimer of five proteins: c-Rel, RelA (p65), RelB, p50, and p52. Under unstimulated conditions, NF-B resides in the cytoplasm by forming complexes with inhibitory B (IB) (27). Proinflammatory stimuli, such as TNF- and lipopolysaccharide (LPS), induce signal cascades through their cognate receptors, TNFR and Toll-like receptor 4 (TLR4), to activate IB kinase (IKK) signalsome and subsequently NF-B that is required for innate and adaptive immune responses to pathogens (13, 38). IKK signalsome is composed of at least two kinases, IKK and -?, and a regulatory factor, IKK (26, 36). Among these proteins, IKK? is known as a major kinase and IKK is required for the full activation of IKK? upon proinflammatory stimulation (41-43, 56, 57, 59, 66, 78). Treatment of cells with TNF- results in IKK signalsome recruitment to the type 1 TNF- receptor (TNFR1), which induces phosphorylation at key residues in the activation loop of the kinase domain of IKK? to be an active kinase (21). Activated IKK signalsome phosphorylates IB, which subsequently undergoes ubiquitination and degradation, liberating NF-B to translocate into the nucleus and activate target genes (37).
NF-B influences gene expression of cyclooxygenase-2 (COX-2) along with other transcription factors, including CREB and C/EBP-? (15, 20, 33, 34, 48, 50, 63, 67, 72, 75). Accordingly, proinflammatory stimuli induce the expression of COX-2 to catalyze the production of prostaglandins, which promote inflammation through a variety of mechanisms.
Our hypothesis is that HCV evades innate immunity by directly infecting immune cells and altering gene expression of key inflammatory molecules. Here we tested whether the HCV core protein affects NF-B activity and COX-2 production in macrophages. We demonstrate that the HCV core protein interacts with IKK?, suppresses its kinase activity, and interferes with nuclear translocation of IKK?, which are correlated with inhibition of NF-B activity. Furthermore, we show that the HCV core protein suppresses NF-B-dependent COX-2 expression. These data indicate that the HCV core protein interrupts diverse IKK? functions, which results in blunted COX-2 expression.
MATERIALS AND METHODS
Cell lines. Murine macrophage cell line RAW 264.7, murine alveolar macrophage cell line MH-S, and HEK 293 and HeLa cells were all obtained from the American Type Culture Collection (Rockville, MD). Cell lines were maintained according to the recommendations of the American Type Culture Collection.
Plasmids. The coding sequence of HCV core (Hutchinson strain, genotype 1a) was inserted into the polylinker site of pCI:neo (Promega). The resultant construct, named pCI-HCV core, was engineered to express the 192-amino-acid-long HCV core protein. To generate a construct that encodes FLAG-tagged HCV core, pCI-HCV core was digested with restriction enzymes NheI and EcoRI (New England BioLabs) and inserted with a linker containing the FLAG sequence. The coding sequence of HCV core and E1 (Hutchinson strain, genotype 1a) was amplified by Pfu polymerase (Stratagene). The forward primer binds to the first 20 nucleotides of the 5' end of the HCV core sequence and contains NheI restriction site, Kozak, and FLAG sequences (5'-GCGGCTAGCCACCATGGACTACAAAGACGATGACGATAAAGGGATGAGCACGAATCCTAAACC-3'; underlined is the core sequence). The reverse primer binds to 18 nucleotides of the 3' end of the HCV E1 sequence and contains HindIII (5'-GCAAGCTTGCCGGCAAATAGCAGCAG-3'). The PCR product was digested with restriction enzymes NheI and HindIII (New England BioLabs) and inserted into the corresponding sites of pcDNA3(-)-Myc/His (Invitrogen). Candidate clones encoding HCV core and E1 were analyzed by sequencing (Vanderbilt DNA Sequencing Core Facility). Expression of the FLAG-tagged HCV core and Myc-tagged protein was determined by Western blotting with M2 antibody (Sigma) and anti-Myc antibody (Santa Cruz Biotechnology). Plasmids encoding a FLAG-tagged IKK and IKK? were kindly provided by R. Carter (Vanderbilt University) and F. Mercurio (Signal Research Division, Celgene Corp., New Jersey), respectively, and a plasmid expressing T7-tagged NEMO/IKK was a gift from E. S. Alnemri (Thomas Jefferson University). A plasmid encoding superrepressor IBS32,36A was a gift from K. Pennington (Vanderbilt University). The luciferase reporter plasmid NF-B-Luc was purchased from Stratagene (La Jolla, CA).
Plasmid transfection, LPS treatment, and luciferase assay. Cells were transfected with Superfect (QIAGEN), as specified by the manufacturer. Each transfection was normalized with appropriate empty vector plasmids. At 48 h posttransfection, transfected cells were harvested for the experiment. For the LPS treatment, after incubation for 14 to 16 h in a 37°C, CO2 incubator, transfected cells were further maintained without serum overnight and subsequently treated with LPS (1 μg/ml in phosphate-buffered saline; Sigma) for 4 h to induce COX-2 expression. For the luciferase assay, cells were cotransfected with luciferase reporter plasmids NF-B-Luc and tk-Renilla Luc. Luciferase activity was measured by a dual-luciferase assay system (Promega) according to the protocol recommended by the manufacturer. NF-B-mediated luciferase activity was normalized with Renilla luciferase activity. The luciferase assay was done in triplicate.
Viruses and infection. Adenoviruses encoding IKK?, superrepressor IkBS32,36A, and ?-galactosidase (?-Gal) were described previously (58). Viruses were propagated and virus titer was determined by standard protocols. RAW 264.7 cells were infected with adenoviruses at multiplicity of infection of 0.2 for 1 h. After inocula were removed, cells were placed in fresh medium and incubated overnight at 37°C in a CO2 incubator. At 24 h postinfection, cells were harvested for the experiment.
Immunoprecipitation and Western blotting. Total cell lysate was prepared with RIPA (radioimmunoprecipitation assay) cell lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1% sodium orthovanadate, 1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) supplemented with phenylmethylsulfonyl fluoride (1 mM), aprotonin (2 μg/ml), leupeptin (2 μg/ml), and soybean trypsin inhibitor (37.5 μg/ml). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Specific bands were revealed using enhanced chemiluminescence (ECL plus; Amersham). For immunoprecipitation analysis, 1 to 2 μg of appropriate antibodies was added to precleared cell lysate that was normalized by protein content and incubated overnight at 4°C. Immune complexes were captured with 30 μl of protein A-Sepharose (Zymed) for 30 min at 4°C and washed five times with RIPA buffer. Antibodies used in this study were as follows: polyclonal anti-murine COX-2 (Cayman Chemical); monoclonal anti-FLAG M2 antibody (Sigma); polyclonal anti-IKK/? and anti-IKK (Santa Cruz Biotechnology); monoclonal anti-IKK and -? (PharMingen); monoclonal anti-HCV core antibody (Affinity Bioreagents, Inc.).
IKK assay. Transfected HeLa cells were treated with 10 ng/ml of TNF- (R&D Systems Inc.) for 15 min before lysis. Cytoplasmic extracts from HeLa or HEK 293 cells were prepared as described previously (49). To immunoprecipitate IKK signalsome, ELB buffer (49) was added to the prepared cytoplasmic extract. Kinase activity was measured in a reaction buffer containing 10 mM HEPES (pH 7.4), 5 mM MgCl2, 1 mM MnCl2, ATP (10 μM), [-32P]ATP (5 μCi), and recombinant glutathione S-transferase (GST)-IB, GST protein fused to amino acids 1 to 54 of IB, at 30°C for 30 min. Radiolabeled products were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and revealed by autoradiography.
Preparation of cytoplasmic and nuclear fractions. Cytoplasmic and nuclear fractions were prepared as described elsewhere (9). In brief, cells were washed three times with ice-cold phosphate-buffered saline and added to hypotonic buffer (10 mM HEPES [pH 7.9], 5 mM KCl, 1.5 mM MgCl2, 1 mM NaF, and 1 mM Na3VO4) supplemented with 1 mM dithiothreitol, phenylmethylsulfonyl fluoride (1 mM), aprotonin (2 μg/ml), leupeptin (2 μg/ml), and soybean trypsin inhibitor (37.5 μg/ml). After lysis for 15 min on ice, the cytoplasmic fraction was prepared by centrifugation at 3,000 rpm for 5 min. The cytoplasmic fraction was cleared by centrifugation at 13,000 rpm for 15 min before further analysis. To prepare the nuclear fraction, the cell pellet generated after initial centrifugation was washed with the hypotonic buffer three times and suspended in high-salt buffer (20 mM HEPES [pH 7.9], 1.5 mM MgCl2, 0.2 mM EDTA [pH 8.0], 420 mM NaCl, 25% glycerol [vol/vol], 50 mM ?-glycerophosphate, 1 mM NaF, and 1 mM Na3VO4) supplemented with 1 mM dithiothreitol, phenylmethylsulfonyl fluoride (1 mM), aprotonin (2 μg/ml), leupeptin (2 μg/ml), and soybean trypsin inhibitor (37.5 μg/ml). The suspended cell pellet was incubated for 30 min on ice with occasional vortexing, and the nuclear fraction was collected after centrifugation at 13,000 rpm for 10 min. Protein content from each fraction was quantified by the Bradford assay (Bio-Rad) as specified by the manufacturer to ensure equal loading.
Statistical analysis. For comparison among groups, paired or unpaired t tests and one-way analysis of variance tests were used (with the assistance of InStat; Graphpad Software, Inc., San Diego, CA). P values of <0.05 are considered significant.
RESULTS
HCV core suppresses NF-B activity in LPS-treated macrophages. HCV core differentially modulates NF-B activity, depending on the subtype of HCV (54). To examine whether the HCV core used in this study (genotype 1a) interferes with NF-B activity in LPS-treated macrophages, RAW 264.7, a murine macrophage cell line, and MH-S, a murine alveolar macrophage cell line, were transfected with a plasmid encoding HCV core along with the NF-B-luciferase reporter construct in the presence or absence of LPS. The results in Fig. 1 showed that HCV core, by itself, did not activate NF-B but rather suppressed NF-B activity elicited by LPS treatment of RAW 264.7 (Fig. 1A) and MH-S (Fig. 1B) cells. A similar experiment using TNF- produced similar results (data not shown). These results demonstrate that HCV core suppresses NF-B activity in macrophages.
HCV core inhibits IKK signalsome kinase activity. To investigate the mechanism by which HCV core inhibits NF-B activity, we tested whether HCV core interferes with IKK kinase activity. For these studies, we employed HeLa and 293 cells because of the ability to achieve high transfection efficiencies in these cell lines. First, to test whether these cell lines were able to recapitulate the results shown in Fig. 1, we performed a similar experiment. As shown in Fig. 2A, when HeLa cells were treated with TNF-, NF-B-mediated transcriptional activity was increased (lanes 1 and 2). However, the presence of HCV core suppressed NF-B activity elicited by TNF- (lanes 2 and 4). Similarly, HCV core suppressed NF-B activated by TNF- in HEK 293 cells (Fig. 2B, lanes 2 and 3). These results suggest that HCV core also represses NF-B activity in these cell lines.
We tested whether coexpression of HCV core and E1 alters the effect of HCV core on NF-B activity. HeLa cells were transfected with a plasmid encoding HCV core and E1 along with the NF-B reporter construct and treated with TNF-. As shown in Fig. 2C, HCV core was able to suppress NF-B activity elicited by TNF- in the presence of HCV E1 (lane 4). A similar result was obtained in HEK 293 cells (data not shown). These results indicate that suppression of NF-B activity by HCV core is not significantly affected by E1.
Next, we tested whether HCV core interferes with IKK signalsome kinase activity. HeLa cells were transfected with increasing amounts of a plasmid encoding HCV core and treated with TNF- (10 ng/ml) for 15 min to induce IKK signalsome activity. The IKK signalsome was immunoprecipitated with anti-IKK? antibody, extensively washed, and incubated with GST-IB and [-32P]ATP for kinase assays. As shown in Fig. 3A, HCV core reduced IKK kinase activity elicited by TNF- treatment in a dose-dependent fashion.
Since IKK? is known as a major kinase in inflammatory gene expression and overexpression of IKK? results in kinase activity that is markedly increased by IKK (24), we examined whether HCV core inhibits IKK? kinase activity in the absence of an activating stimulus (Fig. 3B). HEK 293 cells were transfected with plasmids expressing FLAG-tagged IKK? and T7-tagged IKK in the presence or absence of HCV core. For IKK? kinase assays, IKK? was precipitated with M2 antibody. As shown in Fig. 3B, IKK? kinase activity was markedly enhanced by IKK (lane 2 compared to lane 1); however, this was strongly suppressed by HCV core (lane 3). Western blot analysis also revealed that the phosphorylated form of IKK?, an indicator of kinase activation, was induced by overexpression of IKK. The phosphorylation of IKK? was blocked by ectopic expression of the HCV core protein (middle panel, lanes 2 and 3). These results indicate that HCV core specifically suppresses IKK? kinase activity.
HCV core physically interacts with IKK?. To understand how the HCV core protein suppresses IKK? kinase activity, we examined the physical interaction between the two proteins by immunoprecipitation assay. HeLa cells were transfected with either an empty vector or a plasmid encoding FLAG-tagged HCV core. After cell lysis, the cytoplasmic fraction was incubated with M2 antibody to capture FLAG-tagged HCV core, and the immune complex was analyzed by SDS-PAGE and Western blotting. First, to detect any subunits of IKK signalsome interacting with the HCV core protein, the membrane was incubated with a mixture of anti-IKK, -IKK?, and -IKK antibodies. As shown in Fig. 4A, the HCV core protein coprecipitated with IKK and/or IKK? but not with IKK. To identify the precipitated band, coimmunoprecipitates of the HCV core protein were further analyzed by Western blotting with IKK and IKK? antibodies. The result in Fig. 4B indicated that IKK? but not IKK coimmunoprecipitated with the HCV core protein.
The HCV core interferes with nuclear localization of IKK?. A recent discovery that IKK and -? are located not only in the cytoplasm but also in the nucleus suggests additional functions of IKK signalsome in regulation of gene expression (12). Indeed, IKK is recruited to the NF-B-binding DNA element via physical interaction with CREB-binding protein, which enhances target gene expression (7, 76). IKK is also directly involved in transcription regulation by inhibiting the interaction between IKK and CREB-binding protein (71). Therefore, we investigated the possibility that the HCV core protein affects IKK? activity by interfering with nuclear-cytoplasmic partitioning of IKK?.
To test whether HCV core selectively interferes with IKK? partitioning within a cell, HEK 293 cells were transfected with plasmids encoding FLAG-tagged IKK and IKK? along with the plasmid encoding HCV core. To determine partitioning of IKK and IKK?, cytoplasmic and nuclear fractions were prepared and analyzed by Western blotting with M2 antibody to reveal IKK and -?. As shown in Fig. 5A, while the majority of IKK resides in the cytoplasm, IKK? was detected in both cytoplasmic and nuclear fractions (lanes 1, 2, 5, and 6). However, the presence of the HCV core protein decreased localization of IKK? in the nuclear fraction (lanes 6 and 8), while not significantly affecting the amount of cytoplasmic IKK?. To ensure equal loading of nuclear proteins, the membrane was stripped and probed with anti-YY1 antibody (8, 29, 46).
To further confirm this observation, a similar experiment was conducted to determine the distribution of endogenous IKK? (Fig. 5B). HEK 293 cells were transfected with increasing amounts of the HCV core-encoding plasmid. Transfected cells were treated with 10 μg/ml of TNF- to examine a possible effect on distribution of endogenous IKK?. Western blotting analysis in Fig. 5B showed that distribution of IKK? was not significantly affected by TNF- treatment (compare lanes 1 and 2 and lanes 5 and 6), as indicated in another study (7). However, the HCV core protein reduced nuclear localization of IKK? (lanes 6, 7, and 8). To ensure equal loading of nuclear protein, the membrane was treated as for Fig. 5A to reveal nuclear protein YY1. Taken together, these results indicate that the HCV core protein blocks IKK? shuttling to the nucleus.
NF-B activation elicited by IKK? is sufficient to induce COX-2. Involvement of NF-B in COX-2 expression is well documented in a variety of cell types (15, 20, 77).
To define the relationship between NF-B activation and COX-2 expression in RAW 264.7 cells, we treated the cells with endotoxin and measured COX-2 expression and NF-B activation by Western blot analysis. As indicated in Fig. 6A, COX-2 protein production increased in response to treatment with LPS, and this was associated with degradation of cytoplasmic IB and the appearance of immunoreactive RelA in the nucleus (Fig. 6B). These data show that the macrophage cell line treated with LPS results in both activation of NF-B and COX-2 protein production.
To assess the role of NF-B in COX-2 expression in macrophages without ambiguity associated with cross talk by inflammatory signals, NF-B was activated in a highly specific manner through ectopic expression of IKK? (26), and resultant COX-2 expression was analyzed by Western blotting in two separate murine macrophage cell lines, RAW 264.7 and MH-S. As shown in Fig. 7, ectopic expression of IKK? increases COX-2 protein production in both macrophage cell lines in a dose-dependent fashion. These data indicate that NF-B activation by ectopic expression of IKK? is sufficient to induce COX-2 expression.
To assess the specificity of this response, we examined whether superrepressor IkBS32,36A suppresses COX-2 expression induced by IKK?. First, RAW 264.7 cells were transfected with an NF-B luciferase reporter construct along with the IKK?-expressing plasmid in the absence or presence of the superrepressor-expressing plasmid. As shown in Fig. 8A, transfection of the superrepressor IBS32,36A markedly inhibits NF-B-mediated luciferase activity induced by IKK?. Next, to test whether COX-2 protein production induced by IKK? is suppressed by IBS32,36A, RAW 264.7 cells were infected with replication-deficient adenoviruses encoding kinase-active IKK?, IBS32,36A, or ?-Gal. Western blot analysis in Fig. 8B shows that infection with IKK?-expressing adenovirus induced COX-2 expression, while infection with ?-Gal-expressing adenovirus failed to do so. Coinfection with two adenoviruses encoding IKK? and IBS32,36A blunted the production of COX-2. Taken together, these data demonstrate that selective NF-B activation results in COX-2 expression in macrophage cell lines.
HCV core suppresses COX-2 expression induced by LPS. Finally, we examined whether the inhibitory effect of HCV core on NF-B activity leads to down regulation of COX-2 expression. RAW 264.7 cells were transfected with either an empty host plasmid or a plasmid expressing the HCV core protein. Subsequently, transfected cells were treated with LPS to induce COX-2. As indicated in Fig. 9, HCV core protein, by itself, did not induce COX-2 expression (lanes 3 and 4). In contrast, HCV core protein strongly suppressed COX-2 expression induced by LPS treatment (lanes 5 and 6).
DISCUSSION
Primary HCV infection evades protective immunity and establishes a chronic infection (4, 16). Chronic HCV-infected patients have reduced Th1-type CD4+ T-cell activity and increased Th2-type CD4+ T-cell activity (16, 23). Some chronic hepatitis C patients treated with alpha interferon (IFN-) (14) and ribavirin show enhanced Th1 CD4+ T-cell effector function with improvement of the medical condition (32, 65, 69). An HCV murine model also showed the compromised production of Th1 cytokines, such as interleukin-2 (IL-2) and IFN-, by HCV core (39). Similarly, there is a decreased Th1 cytokine production in transgenic mice that express HCV core (62). These data, together, suggest that HCV evades the host immune response through a mechanism that is mediated by HCV core. Recent experimental evidence shows that HCV infection occurs with a broad cellular tropism encompassing not only hepatocyte cells but also immune cells, such as B and T lymphocytes and monocytes (1). However, the impact of HCV on infected immune cells is not understood. In this study, we addressed this issue by examining an inhibitory effect of HCV core on NF-B, a key transcription factor in innate and adaptive immunity. Our data showed that, through inhibition of IKK kinase activity, HCV core hinders NF-B activity, which results in blunted expression of an important inflammatory protein, COX-2, in macrophages.
Along with constitutively expressed COX-1, COX-2 metabolizes arachidonic acid to prostaglandin H2, which is further metabolized to generate various prostanoids (24). Prostanoids comprise three groups: prostaglandins, such as D2 (PGD2), E2 (PGE2), and F2 (PGF2); prostacylin, such as PGI2; and thromboxane A2. In general, prostanoids are considered to promote inflammation by inducing vasodilation and increasing vascular permeability and cellular migration to the site of inflammation (22, 73, 74). Notably, prostanoids are also important modulators of adaptive immunity (28, 30); however, effects of prostanoids on immunity are complex, and the net result depends on the surrounding milieu. For example, PGE2 promotes dendritic cell maturation and migration to lymphoid organs in peripheral tissue, while it suppresses T-cell activation by dendritic cells in lymphoid organs (28). In the case of PGD2, while enhancing the Th2-type immune response, PGD2 and its derivative cyclopentenone prostaglandins are known to have anti-inflammatory activities by inhibiting NF-B, inducing apoptosis of macrophages and neutrophils in inflammatory lesions, and activating anti-inflammatory transcription factor PPAR- (64). Other prostanoids, such as PGI2 and thromboxane A2, also increase T-cell functions, and PGI2 enhances natural killer (NK) cell functions (19, 35, 68, 70). Nevertheless, these results clearly illustrate that prostanoids are implicated in innate and adaptive immunity.
It is not well documented that there is a relationship between HCV infection and COX-2 expression that might influence HCV pathogenesis. As yet, combined treatment with IFN- and ribavirin is the popular measure to control hepatitis C, albeit with limited success. A report that IFN- increased PGE2 production suggested that elevated PGE2 might be attributed to unresponsiveness to IFN- treatment, given the immunosuppressive nature of PGE2 (6). Accordingly, IFN- signaling can be enhanced by indomethacin, one of the nonsteroidal anti-inflammatory drugs (NSAIDs) that blocks prostanoids production by inhibiting COX-1 and COX-2 activities (25). However, combined treatment of HCV patients with NSAIDs and IFN- has shown limited success and is less effective than treatment with ribavirin (5). This result might be due to complex effects of prostanoids on host immunity. Interestingly, recent studies have shown that arachidonic acid is able to suppress HCV genomic replication and has a beneficial effect on HCV-infected patients (40, 51). Although it is not understood how arachidonic acid affects HCV, it is conceivable that impaired arachidonic acid metabolism might be favorable for HCV survival.
Our data implicate HCV in COX-2 expression. Although additional studies are apparently required to understand the biological significance of deregulated COX-2 expression in HCV-infected cells, possible roles of COX-2 in HCV pathogenesis can be hypothesized. It is possible that HCV avoids unwanted stimulation of adaptive immune responses against itself by down regulating COX-2 expression in HCV-infected antigen-presenting cells, such as macrophages and dendritic cells. Another possibility is that, by down regulating COX-2 expression, HCV-infected immune effector cells avoid lysis by NK cells and/or apoptosis caused by cyclopentenone 15d-PGJ2, which is derived from PGD2 during inflammatory responses. Lastly, HCV avoids evoking antiviral activities of prostanoids by suppressing COX-2 expression. Although a precise mechanism is not known, PGA and PGJ series prostaglandins have been reported to have antiviral activities against various DNA and RNA viruses (64). Recently, PGI2 was reported to have antiviral activity against respiratory syncytial virus in a respiratory syncytial virus-induced disease mouse model (31). Similarly, in a vaccinia virus-induced disease mouse model, PGI2-treated mice showed enhanced survival, whereas NSAIDs-treated mice succumbed to virus-induced mortality (80). Therefore, impaired COX-2 expression could be attributable to host immune evasion by HCV, favoring persistent HCV infection.
COX-2 expression is regulated by diverse transcription factors that include NF-B, CREB, and C/EBP-?. However, the relative importance of these pathways is not clear and, more importantly, the impact of NF-B on COX-2 expression in macrophages is controversial. Our results show that COX-2 is induced by selective activation of NF-B alone and is suppressed by expression of superrepressor IB, suggesting that NF-B activation is sufficient for COX-2 expression.
Our data in Fig. 8 show that COX-2 induction by LPS is substantially inhibited by HCV core. Given the complexity of cox-2 transcriptional regulation and the diverse transcriptional modulating function of HCV core, the possibility that HCV core also influences function of other transcription factors that are involved in COX-2 expression cannot be excluded. It is also possible that the significant decrease of COX-2 is due to a direct interruption of TNF- or other cytokine production, via NF-B suppression by HCV core, which otherwise contributes to COX-2 expression in an autocrine fashion, as LPS treatment induces TNF- and other cytokines that, along with TLR4 signaling, enhance COX-2 expression (11). Finally, it is conceivable that HCV core could alter the half-life or increase the turnover rate of COX-2 protein, although transcription of the cox-2 gene is closely related to the level of COX-2 protein expression (M. D. Bryer, personal communication), and the half-life of COX-2 mRNA is significantly prolonged by LPS treatment (10).
NF-B is either activated or suppressed by HCV core, depending on subtype. For example, while the core protein of HCV type 1b activates NF-B activity, that of HCV type 1a (Hutchinson strain) suppresses NF-B activity (54). A mutagenesis study showed that a single amino acid substitution in the core protein of HCV type 1a converting either from Lys at 9 (K9) to Arg or from Asn at 11 (N11) to Thr is sufficient to relieve its inhibitory function (54). However, it is not understood how these amino acid residues affect modulation of NF-B activity.
Previously, it was proposed that activation of NF-B by type 1b HCV core is related to IKK?, although a direct biochemical interaction between the two proteins and direct functional interference by the HCV core protein were not presented (79). In the present study, we report evidence that type 1a HCV core protein biochemically interacts with IKK? and inhibits IKK kinase activity (Fig. 6A and 7). Although we do not fully understand the mechanism that, unlike type 1a HCV core, type 1b HCV core activates IKK signalsome and NF-B activity, our data raise the possibility that a capability of HCV core to physically interact with IKK? might determine differential regulation of NF-B activity. It is possible that the type 1a HCV core protein sequesters IKK? and prevents formation of a fully functional IKK signalsome, which impairs NF-B signaling. Alternatively, it is possible that the HCV core protein interferes with stoichiometric interactions between IKK? and, subsequently, other subunits of IKK signalsome. More importantly, given that a single amino acid change at either K9 or N11 relieves inhibitory functions of the HCV core protein type 1a, it is conceivable that microenvironmental changes such as electric charge or physical hindrance affect interactions between IKK? and its substrates.
Another observation in our study that might lead to understanding of the mechanism by which HCV core suppresses IKK activity is the disruption of cytoplasmic-nuclear partitioning of IKK?. As shown in Fig. 4, either transfected or endogenous IKK-? is located in both cytoplasmic and nuclear fractions; however, the HCV core protein selectively disturbs the nuclear localization of IKK-?. In similar experiments to measure cellular distributions of IKK and IKK, HCV core did not affect the distributions of these two subunits of the IKK signalsome (data not shown). The functional significance of nuclear IKK? and the biological significance of blocked translocation of IKK? by the HCV core protein are areas of intense investigation.
In summary, we studied the inhibitory function of HCV core on NF-B activity in macrophages. Our data indicate that inhibition of NF-B activity by the HCV core protein might be related to its physical interaction with and interrupted nuclear localization of IKK?. Furthermore, our data show that COX-2 expression is induced by NF-B activation and suppressed by the HCV core protein. These results suggest the possibility that HCV-infected macrophages have impaired immune responses at least in part due to impaired COX-2 expression.
ACKNOWLEDGMENTS
This work was supported by the Department of Veterans Affairs and National Institutes of Health grants AI057591 (Y.S.H.), HL075557 (J.W.C.), and HL061419 (T.S.B.) and program project HL066196.
We thank Tamara Lasakow for editorial assistance with the manuscript.
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