The Hepatitis C Virus Core Protein Modulates T Cell Responses by Inducing Spontaneous and Altering T-cell Receptor-triggered Ca2+ Oscillations*
Anders Bergqvist
¶,
Sara Sundström
,
Lina Y. Dimberg
,
Erik Gylfe || and
Maria G. Masucci
From the
Microbiology and Tumor Biology Centre, Karolinska Institutet, SE-17177 Stockholm, Sweden,
Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Centre, SE-75237 Uppsala, Sweden,
|| Department of Medical Cell Biology, Uppsala University, Biomedical Centre, SE-75237 Uppsala, Sweden
Received for publication, January 8, 2003
, and in revised form, March 5, 2003.
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ABSTRACT
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Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infection, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Expression of the HCV core (C) protein modulates transcription of the IL-2 promoter in T lymphocytes by activating the nuclear factor of activated T lymphocyte (NFAT) pathway. Here we report on the effect of HCV C on Ca2+ signaling, which is essential for activation of NFAT. Expression of HCV C correlated with increased levels of cytosolic Ca2+ and spontaneous Ca2+ oscillations in transfected Jurkat cells. Triggering of the T-cell receptor induced a prolonged Ca2+ response characterized by vigorous high frequent oscillations in a high proportion of the responding cells. This was associated with decreased sizes and accelerated emptying of the intracellular calcium stores. The effect of HCV C on calcium mobilization was not dependent on phospholipase C-
1 (PLC-
) activity or increased inositol 1,4,5-trisphosphate (IP3) production and did not require functional IP3 receptors, suggesting that insertion of the viral protein in the endoplasmic reticulum membrane may be sufficient to promote Ca2+ leakage with dramatic downstream consequences on the magnitude and duration of the response. Our data suggest that expression of HCV C in infected T lymphocytes may contribute to the establishment of persistent infections by inducing Ca2+ oscillations that regulate both the efficacy and information content of Ca2+ signals and are ultimately responsible for induction of gene expression and functional differentiation.
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INTRODUCTION
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Hepatitis C virus (HCV)1 is the major cause of non-A, non-B hepatitis (1). Characteristic features of HCV infection include a high incidence of persistent infection resulting in chronic hepatitis, which is a strong risk factor for liver cirrhosis and hepatocellular carcinoma. Several models have been proposed to explain the failure to establish an efficient immune control that eradicates the infection. A favored hypothesis is that escape from immune surveillance is promoted by the rapid emergence of viral variants. Indeed, during the course of chronic infections, antibodies recognizing the hypervariable region 1 of the viral E2 envelope protein undergo changes in their epitope specificities, suggesting the selection of variants with an enhanced ability to persist in the host (2).
Recent studies have begun to uncover immune response correlates of HCV clearance versus persistence. Overall, these data suggest that virus clearance is associated with the activation of Th2 responses directed against specific viral antigens (3) and with a strong early activation of CD8+ cytotoxic T cell responses against multiple HCV epitopes (4). Robust humoral responses do not generally correlate with virus clearance, and in acute resolvers HCV-specific antibodies are often transient and disappear (5, 6). These findings indicate that a Th1-type rather than a Th2-type response may be beneficial for controlling and clearing HCV infection. Modulation of T cell responses by alteration of the balance between Th1 and Th2 cytokines is the hallmark of many persistent infections and is often associated with the expression of specific viral products (7); such viral factors have not been identified in HCV.
The HCV genome is translated into a polyprotein of about 3000 amino acids that is cleaved by cellular and viral proteinases into three structural proteins and at least six non-structural proteins (8). At least three of the HCV gene products have been suggested to have immunomodulatory functions. The structural protein E2 (9) and non-structural protein NS5A (10, 11) appear to modulate interferon resistance by interacting with the interferon-inducible, double-stranded, RNA-dependent protein kinase R (PKR). In addition, the HCV core (C) protein was recently shown to bind to certain members of the tumor necrosis factor (TNF) receptor superfamily and modulate the sensitivity to TNF-
in some cell types (12, 13, 14, 15). Furthermore, in vitro (16, 17, 18) and some in vivo (19, 20, 21) studies indicate that HCV may replicate in B- and T-lymphocytes suggesting that the activity of immunocompetent cells may be directly influenced by virus infection.
We have recently reported that transient expression of HCV C in human T cells activates the IL-2 promoter by stimulating the nuclear factor of activated T cell (NFAT) pathway (22). This activation was sensitive to Ca2+ deprivation and could be abolished by removal of the C-terminal domain that targets HCV C to the endoplasmic reticulum (ER). The ER is the main storage site for intracellular Ca2+, and mobilization of Ca2+ from this store is an essential triggering signal for different downstream events, including specific gene activation (23). One of the main pathways for mobilization of Ca2+ is controlled by inositol 1,4,5-trisphosphate (IP3)-gated channels located in the ER membrane (24). IP3 is generated by phospholipase C-
1 (PLC
), which is activated by phosphorylation. In T cells, this is accomplished by cross-linking of the T-cell receptor (TCR) complex and subsequent induction of a tyrosine kinase cascade (25). Depletion of intracellular calcium stores results in calcium entry across the plasma membrane via a class of store-operated channels named Ca2+ release-activated Ca2+ (CRAC) channels (26, 27). In the present investigation we have studied the effect of HCV C on the mobilization of Ca2+ in T cells exposed to various stimuli. We demonstrate that expression of HCV C promotes accelerated leakage of Ca2+ from intracellular stores, resulting in spontaneous oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) and prolonged TCR-triggered Ca2+ responses that favor selective activation of NFAT.
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EXPERIMENTAL PROCEDURES
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Chemicals and Immunological ReagentsAll chemicals used were of analytical grade. Restriction endonucleases, T4 DNA polymerase, Taq polymerase, and T4 DNA ligase were obtained from MBI Fermentas (Vilnius, Lithuania). Ionomycin, 12-O-tetradecanoylphorbol 13-acetate (TPA), 2-aminoethoxydiphenyl borate (2-APB), thapsigargin, and puromycin were from Sigma Chemical Co. (St. Louis, MO). The anti-CD3 antibody HIT3a was from Pharmingen (San Diego, CA). Horseradish peroxidase-linked anti-immunoglobulins were from Dako A/S (Glostrup, Denmark). Beetle luciferin was purchased from Promega Corp. (Madison, WI). The monoclonal antibody C750 (28) recognizing HCV C was a gift from Dr. Jack Wands (Harvard Medical School, Boston, MA).
Cells, Cell Culture, and PlasmidsThe human T lymphoma line Jurkat, subclone E61 (29), and its derivative J.Cam1 (30) were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS). The plasmid pUHD/HCV1194 designed for stable expression of HCV C was made by cloning complementary DNA from the infectious HCV H77 strain (31, 32) encoding the first 194 amino acids of the polyprotein into the EcoRI site downstream of the tetracycline-response element of pTRE-luc (Clontech). The plasmid pHCVC
81123 encoding mutated core protein with an internal deletion was made by removal of the KpnI-ClaI restriction fragment of pOP/HCV1194 followed by flush-end ligation. The plasmid pOP/HCV1194 is a cytomegalovirus immediate early promoter-driven HCV C expression vector (22). NFAT-luc contains three copies of the NFAT site (286 to 257 of the human IL-2 gene) linked to the human IL-2 promoter (72 to +47) driving luciferase expression (33). The plasmid pPLC-
(H335F+H380F) encodes a catalytically compromised mutant of PLC-
(34). The plasmid pUHD151 (Clontech) encodes the tetracycline-controlled transactivator (tTA) required for activation of the tetracycline-response element in pTRE-luc. The plasmid pSV2Pac confers puromycin resistance (35).
Stably HCV C-expressing sublines of Jurkat cells were established by transfection with the plasmids pUHD/HCV1194, pUHD151, and pSV2Pac at a ratio of 3:3:1. The transfected cells were placed in 96-well plates and selected in the presence of 0.2 µg/ml puromycin. Resistant clones were screened for HCV C expression by Western blotting. Approximately 5 x 104 cells were resuspended in lysis buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1.0% aprotinin, 1.0 mM dithiothreitol), and the extracts were clarified by centrifugation for 10 min. Proteins were resolved on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). HCV C was detected with the mouse monoclonal antibody C750 (ascites diluted 1:2000). Bound secondary antibody was detected by enhanced chemiluminescence (Pierce, Rockford, IL). All the positive clones exhibited constitutive HCV C expression and were resistant to treatment with doxycycline (data not shown).
Measurements of [Ca2+]iAliquots of 2 x 106 cells were loaded with 5 µM of the Ca2+ indicator Fura-2 acetoxymethyl ester during 40 min at 37 °C in 2.5 ml of RPMI medium and then washed and resuspended in 1 ml of HEPES-buffered medium (25 mM HEPES (pH 7.4), 137 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.3 mM CaCl2, and 10 mM glucose). The cells were then transferred to a 1-cm quartz cuvette placed in the thermostatically controlled cuvette holder of a time-sharing multichannel spectrofluorometer. [Ca2+]i was measured as previously described (36) with compensation for spontaneous leakage of the Ca2+ indicator. For single cell analyses, the cells were adhered to 25-mm diameter glass coverslips by centrifugation at 400 x g for 10 min in serum-free RPMI prior to labeling with Fura-2. The coverslips were then used as exchangeable bottoms of an open 0.16-ml chamber thermostatted at 37 °C and perfused at a rate of 0.5 ml/min with the HEPES-buffered medium. The chamber was placed on the stage of a Diaphot microscope (Nikon, Kawasaki, Japan) equipped with an epifluorescence illuminator and a 40x oil immersion fluorescence objective. A monochromator, which was part of a Quanticell 700 imaging system (VisiTech International, Sunderland, UK), provided excitation light flashes at 340 and 380 nm, and the emission was measured at 515 nm using an intensified charge-coupled device camera. Image pairs were taken every 2.5 s, and 340/380 nm ratio images were calculated after subtraction of background images. [Ca2+]i images were calculated according to Grynkiewicz et al. (37) using a dissociation constant for Ca2+-Fura-2 of 224 nM. Statistical significances were determined by either chi-square analysis or two-tailed Student's t test.
Inositol Phosphate AssayJurkat cells and HCV C-expressing sublines were labeled with 2 µCi of myo-[3H]inositol/ml for 20 h in inositol-free RPMI 1640 supplemented with 5% dialyzed FCS. After blocking of phosphatase activity by 20 mM LiCl for 15 min, cells were stimulated for 10 min with the CD3-specific monoclonal antibody HIT3a (1 µg/ml) in inositol-free RPMI supplemented with 0.5% FCS. Cells were then lysed in acidified methanol, and phospholipids were separated from inositol phosphates by extraction with chloroform/water. The water phase was mixed with AG1-X8 resins (Bio-Rad, Hercules, CA), which were washed with 5 mM sodium tetraborate, 60 mM sodium formate. Radiolabeled inositol phosphate species were eluted in 0.1 M formic acid and 1.0 M ammonium formate and then quantified by scintillation counting.
NFAT Promoter Activation AssaysThe effect of HCV C on activation of NFAT was examined in a reporter assay. Approximately 8 x 106 cells were transfected with 5 µg of DNA in the presence of 0.5 mg of DEAE-dextran per milliliter in 1.6 ml of Tris-buffered saline (25 mM Tris-Cl (pH 7.4), 140 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 0.9 mM CaCl2, 0.5 mM MgCl2). Equal amounts of effector plasmid (pOP/HCV1194) and reporter plasmid (NFAT-luc) were used. At 41 h post transfection, the cells were aliquoted and stimulated with either TPA (50 ng/ml) or a mouse monoclonal antibody recognizing CD3 (HIT3a, 1 µg/ml). Seven hours later, the cells were washed in phosphate-buffered saline and lysed in a buffer containing 25 mM Tris phosphate (pH 7.8), 2 mM dithiothreitol, 10% glycerol, and 1% Triton X-100. Luciferase activity was determined in a Luminoscan luminometer (Labsystems, Vantaa, Finland) using beetle luciferin according to the manufacturer's instructions.
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RESULTS
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Expression of HCV C Alters TCR-triggered Ca2+ Responses in Jurkat CellsWe have previously shown that expression of HVC C in Jurkat cells modulates IL-2 gene transcription by inducing constitutive activation of the NFAT-responsive element (22). Because Ca2+ signaling is involved in NFAT activation (38), and membrane localization is required for HCV C activity, we have now asked whether expression of the viral protein may interfere with Ca2+ mobilization from ER stores. To this end, Jurkat cells were transfected with an eukaryotic expression vector encoding the full-length core protein from the HCV strain H77 and clones growing in puromycin-containing medium were screened for expression of HVC C by Western blotting using a specific mouse monoclonal antibody (Fig. 1A). Three clones expressing relatively high levels of HVC C, JHC.d, JHC.g, and JHC.h, were chosen, along with parental Jurkat cells, for analysis of Ca2+ mobilization in response to triggering with anti-CD3 monoclonal antibody (Fig. 1B). Triggering of the TCR complex in the parental Jurkat cells resulted in a rapid [Ca2+]i peak followed by slow return to a sustained suprabasal level corresponding to Ca2+ influx through store-operated channels. The response was dramatically different in HVC C-expressing cells. First, the basal [Ca2+]i was significantly increased in the three HCV C-positive clones (p < 0.001; n = 22). Second, whereas triggering of the TCR induced an early [Ca2+]i peak was comparable or slightly higher than that observed in the control cells, the late sustained response due to store-operated calcium influx was of higher magnitude and significantly prolonged. The response of a transfected Jurkat clone that did not express HCV C was indistinguishable from that of the parental control (data not shown).

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FIG. 1. TCR-triggered [Ca2+]i response in parental and HCV C-expressing Jurkat cells. A, expression of HCV C in clones of Jurkat cells transfected with an HCV C-expressing plasmid. Expression of HCV C in three representative clones: JHC.d, JHC.g, and JHC.h, was detected in Western blots using a specific mouse monoclonal antibody. The parental Jurkat cell line (Jk) was used as negative control. An extract of HeLa cells transiently transfected with pOP/HCV1194 was used as positive control (+). The positions of molecular weight markers run in parallel are indicated to the right. B, effect of stable expression of HCV C on basal and TCR-induced [Ca2+]i mobilization. JHC.d, JHC.g, and JHC.h cells were loaded with the indicator Fura-2, and [Ca2+]i was monitored for 700 s. At time zero the cells were stimulated with 1 µg/ml of a monoclonal antibody against the TCR complex ( -CD3). Representative traces for six (Jk, JHC.d, and JHC.g) and four (JHC.h) experiments are shown.
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HCV C Induces Spontaneous [Ca2+]i Oscillation and Modifies TCR-triggered Oscillations[Ca2+]i oscillations are the most common mode of cellular signaling, and the amplitude and frequency of oscillation regulate the expression of genes driven by proinflammatory transcription factors such as NFAT (39, 40, 41, 42). To investigate the effect of HCV C on [Ca2+] oscillations, the levels of spontaneous and TCR-triggered [Ca2+]i was monitored over time in individual Jurkat and JHC.d cells using digital video imaging techniques. Representative recordings illustrating the behavior of control and HCV C-expressing cells before and after TCR stimulation are shown in Fig. 2, and the results of 306 tests are summarized in Table I. In the absence of TCR triggering, spontaneous low amplitude [Ca2+]i oscillations were observed in a 3-fold higher proportion of the HCV C-expressing cells than in the control cells (p < 0.001; n = 306), which probably explains the higher average levels of resting [Ca2+]i in suspensions of HCV C-expressing cells (compare Figs. 1B and 2). A [Ca2+]i peak was induced by TCR triggering in both Jurkat and JHC.d cells (Fig. 2), but the late response was altered in the HCV C-expressing cells. Although the average [Ca2+]i decreased within 10 min to less than 10% of the initial peak response in control cells, the response was considerably prolonged in JHC.d cells. Irregular oscillations were observed during the declining phase in control cells, whereas in JHC.d cells the sustained phase was characterized by vigorous, high frequency oscillations. These vigorous oscillations, defined as at least four consecutive calcium spikes with a reduction of at least 50% between peak values, were observed in 46% of the JHC.d cells, which is a 5-fold increase compared with controls (p < 0.001; n = 306). Moreover, the average oscillation period was reduced by more than 40% in the JHC.d cells (p < 0.001; n = 18).

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FIG. 2. [Ca2+]i response in individual cells. Control cells (Jk, upper graph) or cells expressing HCV C (JHC.d, lower graph) were loaded with the indicator Fura-2, and [Ca2+]i was monitored for 1400 s. At time zero, cells were stimulated with 1 µg/ml of a monoclonal antibody directed against the TCR-complex ( -CD3). Representative traces for Jurkat and oscillating JHC.d cells are shown.
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Store-operated Ca2+ Channels Are Partially Activated in HCV C-expressing CellsBinding of IP3 to its receptor in the ER membrane depletes this Ca2+ store, which triggers the entry of extracellular calcium via CRAC channels in the plasma membrane. To investigate whether the differences in Ca2+ mobilization may result from activation of CRAC channels in HCV C-expressing cells, we investigated the response of [Ca2+]i to changes in the extracellular Ca2+ concentration. Chelation of extracellular Ca2+ by EGTA had little effect on [Ca2+]i in Jurkat cells (Fig. 3). After reintroduction of the ion, the sarcoendoplasmic reticulum calcium-ATPase (SERCA) pump inhibitor thapsigargin induced a biphasic response with pronounced sustained elevation of [Ca2+]i, which was sensitive to omission of extracellular Ca2+ indicating CRAC channel activation. In contrast, JHC.d cells showed a pronounced [Ca2+]i response to depletion of extracellular Ca2+ under basal conditions (p < 0.001; n = 8) indicating that the high [Ca2+]i observed in the transfectant (see Fig. 2) is at least in part due to CRAC channel activation. In line with this possibility, [Ca2+]i was similar in JHC.d and Jurkat cells kept in Ca2+-deficient medium, and the difference could be restored by normalization of the extracellular Ca2+ concentration. Furthermore, the additional activation of CRAC channels induced by thapsigargin was smaller in HCV C expressing than in control cells (p < 0.001; n = 8).

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FIG. 3. [Ca2+]i response to changes in the extracellular Ca2+ concentration. [Ca2+]i was measured in control cells (Jk) and cells expressing HCV C (JHC.d) as described in the legend to Fig. 1B. Where indicated, EGTA, CaCl2, and thapsigargin corresponding to final concentrations of 2 mM, 2 mM, and 100 nM, respectively, were added. Traces representative for three independent experiments are shown.
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Expression of HCV C Correlates with Decreased Size and Accelerated Depletion of Intracellular Ca2+ StoresBecause the CRAC channels are activated by depletion of calcium from the ER, we studied whether HCV C affects this calcium store. The size of the ER calcium pool was analyzed by thapsigargin-induced, selective depletion of these stores in the absence of extracellular calcium. Under these conditions, the increase of [Ca2+]i is solely a consequence of emptying of the ER stores and the clearance of Ca2+ is ultimately due to outward transport. We found that Ca2+ was cleared from the cytoplasm at the same rate in all cell lines, and we therefore used the time integral of the [Ca2+]i response as an indirect measure of the ER calcium pool size (Fig. 4A). The amount of calcium mobilized by thapsigargin was reduced by about 50% in HCV C-expressing cells as compared with Jurkat control cells (p < 0.001; n = 12). Moreover, the kinetics of Ca2+ efflux was faster in HCV C-positive cells with peak [Ca2+]i being reached at 30 and 55 s, respectively (Fig. 4, p < 0.001, n = 12). To assess whether the accelerated kinetics was a consequence of the smaller Ca2+ pool or reflected an intrinsic difference in calcium permeability, the intracellular stores were partially depleted by prolonged superfusion with Ca2+-deficient medium. Also under these conditions the thapsigargin-induced Ca2+ efflux was more rapid in C-expressing cells (Fig. 4B), indicating that the rate of depletion was unrelated to the pool size. Indeed, the [Ca2+]i peaks occurred with identical delays irrespective of whether the cells had been exposed to Ca2+-deficient medium for 6 (Fig. 4A) or 12 (Fig. 4B) min.

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FIG. 4. Effects of HCV C on thapsigargin-induced mobilization of Ca2+ from the ER. Fura-2-loaded cells were resuspended in Ca2+-deficient medium followed by addition of 2 mM EGTA. After 6 min (A)or 12 min (B) the ER was depleted of Ca2+ by addition of 100 nM thapsigargin. [Ca2+]i was measured as described in the legend to Fig. 1B. Traces representative for six (Jk and JHC.d) and three (JHC.g and JHC.h) independent experiments are shown in A. The traces in B are representative of three experiments.
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Altered Ca2+ Mobilization Is Independent of PLC-
, IP3 Production, and Binding to the IP3 ReceptorStimulation of the TCR complex induces, via a tyrosine kinase cascade, activation of PLC-
, which generates IP3 that is ultimately responsible for Ca2+ mobilization. To investigate whether the effect of HCV C on calcium signaling is dependent on functional PLC-
, its activation by TCR was blocked by expression of PLC-
(H335F+H380F), a lipase-inactive, dominant negative mutant of PLC-
(34). To eliminate the background derived from untransfected cells, Ca2+ signaling was estimated by measuring a downstream target of Ca2+ mobilization, the activity of NFAT. NFAT acts synergistically with AP-1 on composite DNA elements that contain adjacent NFAT and AP-1 binding sites (43). Jurkat cells were co-transfected with pPLC-
(H335F+ H380F), an NFAT-driven luciferase reporter and an HCV C plasmid expressing a core deletion mutant (pHCV
81123) that is hyperactive with respect to induction of NFAT-dependent transcription.2 Transfected cells were stimulated with a CD3-specific antibody and TPA, which activate AP-1 via the Ras/mitogen-activated protein kinase pathway, and luciferase activity was then determined. As shown in Fig. 5, NFATmediated transcription was induced by cross-linking the TCR complex or by expression of HCV C. However, although the TCR-triggered response was prevented by expression of dominant negative mutant PLC-
(Fig. 5A), this did not affect HCV C-induced activation of NFAT (Fig. 5B) confirming that the effect occurs downstream of PLC-
.
To test whether HCV C may induce calcium signaling by stimulating IP3 generation via a PLC-
-independent route, the production of IP3 was compared in HCV C-expressing cells, Jurkat cells, and the p56lck-deficient Jurkat derivative J.Cam1. Cells labeled with myo-[3H]inositol were stimulated with an antibody to the TCR complex, and radiolabeled inositol phosphates were purified and quantified. Cross-linking of the TCR resulted in increased IP3 levels in Jurkat cells, whereas no response was obtained in J.Cam1 cells, as expected (Fig. 6). Comparable levels of IP3 were induced in control and HCV C-expressing cells. However, somewhat surprising, all the HCV C-expressing clones displayed decreased basal IP3 levels in comparison with Jurkat and J.Cam1 cells.

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FIG. 6. Effects of HCV C on the generation of IP3 induced by TCR triggering. Jurkat cells (Jk), a Jurkat mutant defective in TCR-mediated signaling (J.CaM1), and the HCV C-expressing clones (JHC.d, JHC.g, and JHC.h) were loaded with myo-[3H]inositol. Basal levels of intracellular inositol phosphates () were determined in cell lysates by scintillation counting. Where indicated (+), cells were stimulated for 10 min prior to lysis with a monoclonal antibody directed against CD3. Values are mean of duplicates (±S.E.) and are normalized to unstimulated Jurkat cells. One representative experiment of three is shown.
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Because both HCV C and the IP3 receptor are localized within the same cellular compartment, a conceivable hypothesis is that HCV C may cause accelerated emptying of intracellular stores by deregulating the IP3-gated calcium channel. To investigate the function of IP3 receptors, the kinetics of thapsigargin-induced store depletion was analyzed in the presence of the membrane permeable IP3-receptor inhibitor 2-APB (44). In the presence of this inhibitor, the thapsigargin-induced [Ca2+]i peak occurred slightly earlier, and the decay was faster in both Jurkat and JHC.d cells (Fig. 7). However, the Ca2+ efflux from the ER stores was still accelerated in the HCV C-expressing cell line (p < 0.01, n = 8). Thus, the ER leakage observed in HCV C-expressing cells does not appear to be dependent on inappropriate IP3 receptor triggering.

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FIG. 7. Inhibition of the IP3-receptor does not prevent the accelerated depletion of intracellular Ca2+ stores in HCV C-positive cells. Fura-2-loaded cells were resuspended in calcium-free buffer in the presence of 2 mM EGTA. The IP3 receptor was blocked by addition of the inhibitor 2-APB (100 µM), and depletion of the ER Ca2+ stores was induced by addition of thapsigargin (100 nM). [Ca2+]i was measured as described in the legend to Fig. 1B. One representative experiment of four is shown.
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DISCUSSION
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We have previously reported that transient expression of HCV C in human T cells facilitates the activation of the IL-2 promoter by stimulating an NFAT-dependent response (22). We now investigated the mechanism of HCV C-mediated activation of NFAT and found that Jurkat cells stably expressing HCV C are characterized by increased basal levels of cytosolic [Ca2+]i. A closer examination of individual cells suggests that the higher average [Ca2+]i level is probably due to the presence of a high proportion of cells displaying spontaneous [Ca2+]i oscillations. HCV C-expressing cells were much more sensitive to changes in the extracellular Ca2+ concentration than control cells suggesting that the increased spontaneous activity may be due to activation of Ca2+ channels. Because the CRAC channels are activated by mobilization of intracellular calcium stores, we measured how much Ca2+ could be released from these stores by treatment with the intracellular Ca2+-ATPase inhibitor thapsigargin. We found that the intracellular calcium stores were reduced in cells expressing HCV C. Moreover, kinetic analysis revealed accelerated emptying of the intracellular calcium pool. Triggering of the TCR results in generation of IP3, which releases Ca2+ from the ER. Thus, HCV C expression may lead to activation of the IP3-gated channels. Furthermore, because HCV C has been found to interact with certain members of the TNF receptor superfamily, a Ca2+ response may also be triggered by interaction with a surface receptor. However, activation by HCV C expression was neither dependent on PLC-
activity nor associated with increased generation of IP3. In fact, basal IP3 levels were lower in C-expressing cells compared with control, perhaps reflecting an adaptation to altered calcium homeostasis. Moreover, accelerated depletion of internal stores was observed also in the presence of the IP3-receptor inhibitor 2-APB. The difference in calcium pool size was reduced under these conditions, conceivably due to indirect, 2-APB-mediated inhibition of the CRAC channels, because these channels functionally interact with the IP3 receptor (45, 46). Intact CRAC channels and a sustained [Ca2+]i response are essential for activation of NFAT (see below). Indeed, HCV C-mediated activation of NFAT was compromised by 2-APB.2 Taken together, these data indicate that HCV C does not affect upstream signaling and is not dependent on intact IP3 receptors for exerting its effect on Ca2+ signaling.
Ca2+ gradients across the ER and plasma membranes are maintained by the SERCA and plasma membrane calcium-ATPases (PMCA), respectively. An increase in [Ca2+]i causes activation of these pumps, which together with concomitant feedback regulation of CRAC- and IP3-gated channels will result in restoration of the normal calcium concentration. In the case of PMCA, the response to [Ca2+]i is biphasic, comprising an instant component and a delayed activity that is maintained for minutes, even when [Ca2+]i is normalized. The effect of thapsigargin on calcium entry was proportionally reduced in HCV C-expressing cells, perhaps reflecting an adaptation to elevated [Ca2+]i by increased PMCA activity. Alternatively, the expression of CRAC channels may be down-regulated. Because the feedback regulation of both the Ca2+ pumps and Ca2+ channels is highly cooperative, restoration of the normal calcium concentration tends to be delayed and accompanied by oscillations. Thus, a relatively small increase in [Ca2+]i may have dramatic effects on Ca2+ mobilization from the ER and on the probability to induce oscillations.
A major unresolved question in cell signaling is how a messenger like Ca2+ with a large number of downstream targets elicits selective responses. Recently, several studies have indicated that the amplitude and duration of the Ca2+ signals control triggering of specific subsets of calcium-sensitive transcription factors. For instance, NF
B and the c-Jun N-terminal kinase are activated by a short high amplitude [Ca2+]i spike (41, 42). In contrast, NFAT is activated by a Ca2+ signal of relatively low amplitude but of longer duration. Moreover, at relatively low levels of [Ca2+]i, oscillations appear to be more effective than a steady elevation in activating NFAT-dependent transcription. Upon cross-linking of the TCR complex, a transient elevation of [Ca2+]i due to mobilization from intracellular stores is followed by a sustained response corresponding to entry via the CRAC channels. In HCV C-expressing cells, the sustained response was more pronounced and was characterized by vigorous and rapid oscillations. It should be stressed that our previous studies indicated that HCV C expression has no effect on NF
B- or AP1-dependent transcription in Jurkat cells (22), suggesting that the pattern of [Ca2+]i signaling may be important for the type of response.
HCV C is normally associated with the ER membrane via its hydrophobic tail, and this association can be prevented by elimination of the C residues downstream of amino acid 153 (47, 48, 49). Because a mutant lacking the C terminus is unable to activate NFAT, localization to the ER membrane may be a prerequisite for such activation. It is therefore conceivable that HCV C increases Ca2+ leakage from the ER, either by interacting with existing channels, or by altering membrane permeability. Although the increased Ca2+ leakage was neither dependent on intact IP3 receptors nor on the SERCA pump, we cannot exclude the involvement of other membrane receptors, such as the ryanodine receptor type 3, which is regulated by cyclic ADP-ribose and is required for Ca2+ oscillations in T cells (50). Because infection by viruses that assemble on the ER membrane results in overexpression and accumulation of a limited set of structural virus proteins in this compartment, overexpression of HCV C might be expected to cause unspecific perturbation of the ER membrane. However, it seems unlikely that the effect on Ca2+ signaling observed in our experiment was solely a consequence of ectopic overexpression of HCV C, because we were able to isolate a C deletion mutant that failed to activate NFAT despite high level accumulation in the ER.2 The recognition of viral proteins capable of enhancing membrane permeability has lead to the description of a new protein family, the viroporins (51). General features of these membrane-disturbing proteins are the occurrence of an amphipathic
-helix, which often contains cationic amino acids in the hydrophilic face, and the ability to form oligomers. Among the viroporins identified so far, poliovirus 2BC (52), coxsackie B3 virus 2B protein (53), and rotavirus NSP4 (54) are capable of disrupting the intracellular Ca2+ homeostasis in infected cells. In addition to the amphipathic
-helix, a second hydrophobic domain was required for these activities in coxsackie B3 virus 2B. Indeed, both a putative amphipathic
-helix motif and a hydrophobic domain are present downstream of amino acid 150 in the C-terminal region of HCV C. Moreover, HCV C is known to homo-oligomerize, and we are currently exploring whether this effect is a critical factor for disruption of the intracellular Ca2+ homeostasis. Finally, HCV C has also been found to interact with a number of cellular proteins (55), and, although none of them seem to be linked either to the ER or to ion channels, their significance in this context cannot be ruled out.
In conclusion, our results demonstrate that HCV C expression reduces the amounts of calcium in the ER stores by increasing leakage of the ion. The partial store depletion promotes Ca2+ entry via CRAC channels favoring [Ca2+]i oscillations, which specifically activate NFAT. Because NFAT is important for transcription of many cytokine genes, such as IL-2, IL-4, TNF-
, interferon-
, and granulocyte-macrophage colony-stimulating factor, expression of HCV C may have profound effects on cytokine synthesis and thereby modulate the immune response. It should be noted, however, that activation of NFAT alone is not sufficient for cytokine induction. Triggering of the TCR without concomitant stimulation of the CD28 co-receptor results in T cell anergy. Indeed, expression of HCV C was associated with decreased inducibility of IL-2 and interferon-
in heterologous recombinant vaccinia and transgenic mouse models (56, 57). Elevated levels of intracellular [Ca2+]i may also affect other cellular processes relevant for HCV pathogenesis, including apoptosis and oxidative stress, and HCV C expression was shown to modulate both functions (58, 59). Although the biological relevance of our findings in the context of HCV infection remains to be established, further studies are warranted and may contribute to the understanding of HCV pathogenesis.
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FOOTNOTES
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* This work was supported by grants from the Swedish Cancer Society, the Swedish Foundation for Strategic Research, and the Karolinska Institutet (to A. B. and M. G. M.), the Swedish Research Council (contracts 72X-06240 to E. G. and 16X-14228 to A. B.), and the Swedish Society for Medical Research, Åke Wibergs stiftelse, and Magnus Bergvalls stiftelse (to A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
¶ To whom correspondence should be addressed: MTC, Karolinska Institutet, Box 280, Stockholm SE-171 77, Sweden. Tel.: 46-8-728-7149; Fax: 46-8-331-399; E-mail: anders.bergqvist{at}mtc.ki.se.
1 The abbreviations used are: HCV, hepatitis C virus; HVC C, HVC core; TNF, tumor necrosis factor; NFAT, nuclear factor of activated T cell; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; PLC-
, phospholipase C-
1; TCR, T-cell receptor; CRAC, Ca2+ release-activated Ca2+; [Ca2+]i, cytoplasmic Ca2+ concentration; TPA, 12-O-tetradecanoylphorbol 13-acetate; 2-APB, 2-aminoethoxydiphenyl borate; SERCA, sarcoendoplasmic reticulum calcium-ATPase; PMCA, plasma membrane calcium-ATPases; IL-2, interleukin-2; FCS, fetal calf serum. 
2 A. Bergqvist, unpublished observation. 
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ACKNOWLEDGMENTS
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The technical assistance of Linnea Brandt and Andreas Bermsel is gratefully acknowledged. We thank Lars Rönnstrand, Tom Steinberg, and Jack R. Wands for fruitful discussions and for providing antibodies, plasmids, and cell lines; Elaine Vieira for help with single cell Ca2+ measurements; and Seisuke Ota for critical reading of the manuscript.
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