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J Biol Chem, Vol. 274, Issue 43, 30722-30728, October 22, 1999


Protein Kinase C Recognizes the Protein Kinase A-binding Motif of Nonstructural Protein 3 of Hepatitis C Virus*

Peter BorowskiDagger §, Julian Schulze zur Wieschparallel , Kerstin Resch, Hubert Feucht, Rainer Laufs, and Herbert SchmitzDagger

From the Dagger  Abteilung für Virologie, Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Straße 74, 20359 Hamburg, Germany and the  Institut für Medizinische Mikrobiologie und Immunologie, Universitätskrankenhaus Eppendorf, Martinistrabeta e 52, 20246 Hamburg, Germany

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) inhibits the nuclear transport and the enzymatic activity of the catalytic subunit of protein kinase A. This inhibition is mediated by an arginine-rich domain localized between amino acids 1487-1500 of the HCV polyprotein. The data presented here indicate that the arginine-rich domain, when embedded in recombinant fragments of NS3, interacts with the catalytic site of protein kinase C (PKC) and inhibits the phosphorylation mediated by this enzyme in vitro and in vivo. Furthermore, a direct binding of PKC to the NS3 fragments leads to an inhibition of the free shuttling of the kinase between the cytoplasm and the particulate fraction. In contrast, a peptide corresponding to the arginine-rich domain (HCV (1487-1500)), despite also being a PKC inhibitor, did not influence the PKC shuttling process and was transported to the particulate fraction by the translocating kinase upon activation with tetradecanoylphorbol-13-acetate. Using the tetradecanoylphorbol-13-acetate -stimulated respiratory burst of NS3-introduced neutrophils as a model system, we could demonstrate that NS3 is able to block PKC-mediated functions within intact cells. Our data support the possibility that NS3 disrupts the PKC-mediated signal transduction.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In more than 50% of the investigated cases, liver infections with the hepatitis C virus (HCV)1 result in chronic liver disease, which often leads to cirrhosis and hepatocellular carcinoma (1, 2). HCV is a small enveloped RNA virus. Its genome is similarly organized to that of pesti- and flaviviruses (3). The viral genome encodes a polyprotein of 3010 amino acids that is processed by host and viral proteases into core protein, envelope proteins (E1 and E2), and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (4). Although a great amount of data has been accumulated about functional regions in these proteins, relatively little is known about their intracellular targets.

Previously, we have reported that NS3 contains an arginine-rich amino acid sequence: Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 (5) (numbered according to the HCV polyprotein (6)). This sequence is similar to sequences occurring in the regulatory subunit of the cAMP-dependent protein kinase, in protein substrates of this enzyme and in the heat stable inhibitor of PKA (7). In fact, this NS3 protein sequence mediates the inhibition of PKA activity. The catalytic subunit of the kinase directly binds to the viral protein, and consequently the cytoplasm-to-nucleus translocation of the kinase is slowed down (8).

Arginine residues are also known to be recognition motifs of substrates and autoregulatory regions of numerous serine/threonine specific protein kinases (9). This is the reason for overlapping specificities of the kinases with regard to their substrates (10) or to their inhibition mediated by inhibitory peptides resembling their autoinhibitory domains (11).

Recently we have shown that a peptide (HCV (1487-1500)) that reproduces the PKA binding domain of NS3 mentioned above is recognized by another serine/threonine protein kinase, the PKC. The peptide directly interacts with the kinase and serves as an excellent substrate for the enzyme (12). Therefore, it seems likely that the arginine-rich sequence embedded in a larger fragment of the HCV polyprotein interacts with the catalytic domain of PKC, as observed in the case of PKA in vivo (8). By means of these protein-protein interactions, the PKC functions could be blocked or slowed down. These interactions may be a new important aspect of pathogenesis of the HCV infection.

In the present study we investigate the mechanisms by which PKC interacts with NS3 in vitro. Furthermore, we examine certain biological effects controlled by PKC in cells harboring the viral protein. Our results provide the first evidence that NS3 affects PKC functions by a complex mechanism similar to the one that impairs the functions of PKA.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Affinity-purified rat brain PKC isolated according to the method described previously (13) was a generous gift of Dr. Hilz (University of Hamburg). Human HCV-positive antisera against NS3 and NS4 of HCV were kindly provided by Dr. Polywka (University of Hamburg). Peptides were synthesized and purified by high pressure liquid chromatography by Dr. Kullmann (Zentrum für Molekulare Neurobiologie of the University of Hamburg). [gamma -32P]ATP (3000 Ci/mmol) and 125I-labeled protein A (30 mCi/mg) were obtained from Amersham Pharmacia Biotech. N-Succinimidyl [2,3-3H]propionate and 125I were purchased from DuPont. Antibody (MC5) specific for alpha -,beta I- and beta II-PKC-isoforms and all other chemicals were obtained from Sigma. Blood from healthy volunteers was kindly provided by Dr. Roos (Abteilung für Transfusionsmedizin, University of Hamburg).

Cell Cultures, Protein Introduction, and Respiratory Burst-- Highly purified neutrophils (> 95%) were prepared from freshly drawn blood by Ficoll-Hypaque centrifugation, dextrane sedimentation, and hypotonic lysis of red cells (14). Neutrophils were resuspended in complete RPMI 1640 Medium supplemented with 10% fetal calf serum and kept at 37 °C in humified air containing 10% CO2. The introduction of proteins or peptides by protease permeabilization was performed as described previously (15). The persistence of the introduced proteins in neutrophils was determined as follows. The introduced cells were suspended in RPMI 1640 medium, and at regular time intervals aliquots of the cell were harvested for immunoblot analysis with affinity-purified anti-NS3 antiserum. The signals were measured by counting the radioactivity of [125I]protein A-labeled bands. The stability of the introduced peptide was measured with 3H-labeled HCV (1487-1500) (see below). Aliquots of the [3H]HCV (1487-1500)-introduced cells were analyzed by 20% SDS/PAGE. The dried gels were exposed to Kodak BioMax MR film supplemented by intensifying screen (BioMax TranScreen). The parts of the gel containing the peptide were cut out and subjected to scintillation counting. The respiratory burst of neutrophils was analyzed by determing the rate of superoxide production by superoxide dismutase-inhibitable reduction of cytochrome c using visible absorption spectroscopy (14).

Cloning and Expression of the Recombinant HCV Protein-- The HCV proteins HCV polyprotein-(1189-1525) and HCV polyprotein-(1923-2023) were cloned, expressed, and purified as described previously (5, 8). For the expression of HCV polyprotein-(1400-1615), the DNA was polymerase chain reaction-amplified using the sense primer, 5'-TCTGAATCGGCCGGGAGATACTGCTCGG-3', and the antisense primer, 5'-GTGAATTCAAGAAGTGCGACGAAC-3', under standard conditions (16). The polymerase chain reaction product was cloned into the vector pTrxFus downstream of the thioredoxin gene (TRX) and expressed in Escherichia coli. The fusion protein TRX-HCV polyprotein-(1400-1615) was purified by affinity chromatography on aminophenylarsine oxide-agarose as recommended by the manufacturer (Invitrogen). The purification procedure was completed by gel filtration chromatography (HiLoad, Amersham Pharmacia Biotech). The purity of the proteins was verified by silver staining after SDS/PAGE.

Kinase Assays-- The protein kinase C activity was determined according to the method of Hannun and Bell (17) in a mixed micells assay with modifications described previously (5). The kinetic parameters were determined by a nonlinear regression analysis using ENZFITTER (BioSoft), and the graphical analyses were performed with SIGMA PLOT (Jandel Corp.).

Labeling Procedures-- To label proteins with fluorescein isothiocyanate (FITC), equal volumes of the protein solution (5 mg/ml in 50 mM Hepes, pH 7.5) and of FITC (1 mg/ml in 0.5 M NaHCO3/Na2CO3 buffer, pH 7.5) were mixed and incubated for 2 h at room temperature. The modified proteins were isolated with Sephadex G-25 in 20 mM Hepes, pH 7.5, and concentrated with Microcentrifuge filters (10 kDa) (Millipore). The FITC labeling of the GST-HCV polyprotein-(1189-1525) did not affect its binding to PKC in an overlay assay. The 3H-labeling of HCV (1487-1500) was performed with N-succinimidyl [2,3-3H]propionate according to the procedure described by Muller (18). The reaction mixture was applied to a HA-Ultrogel column (approximately 5 mg peptide/1 ml matrix), and the column was washed with 10 mM ammonium acetate, pH 7.8. Bound peptide was eluted with 0.5 M ammonium acetate, pH 9.5, and the fraction was freeze-dried for 24 h. PKC (50 pmol) was radioiodinated in a reaction with Iodogen (1,3,4,6-tetrachloro-3,6-diphenylglycouril) as described by Judd (19). Unbound radioactivity was separated on a NICK Column (Sephadex G-50, Amersham Pharmacia Biotech) pre-equilibrated with TTBS buffer (see below).

Immunoblotting and Binding Assays-- Immunoblot analyses were performed as described by Towbin et al. (20). The transferred proteins were probed with antisera (diluted 1:1000) and then with rabbit anti-mouse or with rabbit anti-human antibodies followed by binding to [125I]protein A. The nitrocellulose was dried and subjected to autoradiography. The protein binding was investigated in an overlay method as described previously (8).

Metabolic Labeling with [32P]Orthophosphate and Phosphorylation of Heat Stable Proteins in Vivo and in Vitro-- The metabolic labeling with [32P]orthophosphate was performed as described previously (8). Briefly, neutrophils (107 cells) were introduced and suspended in RPMI 1640 medium for 4 h as described above. Thereafter the cells were washed and suspended in phosphate-free RPMI 1640 medium supplemented with 2.5% fetal calf serum for 30 min. The medium was replaced by medium containing [32P]orthophosphate (at a final concentration of 0.5 mCi/ml) and incubated at 37 °C for 2 h. During this incubation, TPA treatment (100 nM) was carried out for 5 and 20 min before the end of labeling. The cells were pelleted by centrifugation, washed with cold 150 mM NaCl, and extracted with 100 µl of buffer containing 100 mM Hepes, pH 7.8, 0.5% Triton X-100, 2 mM NaF, 100 µM Na3VO4, 2 mM EDTA, 2 mM EGTA, 5 mM phenylmethylsulfonyl fluoride, 2 mM N-tosyl-L-phenylalanine chloromethyl ketone, 2 mM N-p-tosyl-L-lysine chloromethyl ketone, 1 µg/ml pepstatin, and 50 µg/ml leupeptin for 5 min at 30 °C. The extracts were boiled for 10 min and centrifugated at 25,000 × g for 20 min. The supernatants were mixed with SDS sample buffer and heated at 95 °C for 5 min (21). Phosphoproteins were resolved by SDS/PAGE and visualized by autoradiography. Heat stable proteins serving as PKC substrates in in vitro reactions were partially purified according to the method described by Mahadevan et al. (22) with the following modifications. Cells were washed with cold 150 mM NaCl and resuspended in hypotonic buffer containing 20 mM Hepes, pH 8.0, 2 mM EDTA, 2 mM EGTA, 5 mM phenylmethylsulfonyl fluoride, 2 mM N-p-tosyl-L-lysine chloromethyl ketone, 2 mM N-tosyl-L-phenylalanine chloromethyl ketone, 1 µg/ml pepstatin, and 50 µg/ml leupeptin. The suspension was processed in a Dounce homogenizer (20 strokes of glass) and centrifugated at 1,000 × g for 10 min. The supernatant was centrifugated at 100,000 × g for 30 min, and the resulting pellet was resuspended in the same buffer as mentioned above supplemented with 0.5% (v/v) Triton X-100, boiled for 10 min, and centrifuged again at 25,000 × g for 20 min. The supernatant was used for the in vitro phosphorylation assays. It should be mentioned that p80 present in the preparation showed the same migration pattern in SDS/PAGE as the p80 phosphorylated in intact cells (Ref. 22 and data not shown).

Direct Fluorescence Microscopy-- Introduced neutrophils were plated on coverslips, dried, fixed with acetone for 10 min at 4 °C, air dried, and incubated with Hoechst number 33258 (2'-[4-Hydroxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole) (5 µM in H2O) for 10 min at room temperature. After washing with destilled water the coverslips were placed in glycerol containing 10% (v/v) phosphate-buffered saline, pH 8.5, and 0.1% (w/v) 1,4-phenyldiamin. The cells were viewed with a Zeiss AXIOPLAN microscope equipped with fluorescence illuminator and photographed on Kodak film.

Other Methods-- The cytosolic and particulate fractions of the neutrophils were prepared as described above (22). The protein concentration was measured according to the method of Lowry et al. (23).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Domains of NS3-- The comparison of the amino acid sequence of the NS3 arginine-rich region: Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 with the sequence of the PKC autoregulatory domain revealed a similarity. A synthetic peptide reproducing the sequence above served as an excellent substrate for the kinase and inhibited the kinase activity competitively in respect of other substrates (12). Therefore, we now examined the possibility that NS3 interacts via its arginine-rich region with PKC in vitro and affects PKC-mediated functions in vivo.

To obtain sufficient amounts of HCV proteins, we cloned and expressed the proteins in E. coli. Because the entire NS3 protein was not found to be readily soluble (Ref. 24 and data not shown), we investigated the interaction between NS3 and PKC, using the well characterized C- and N-truncated form of the NS3 protein (HCV polyprotein-(1189-1525)) (5, 8). A second NS3 fragment that also contains the amino acid sequence HCV polyprotein-(1400-1615) was synthesized to ascertain the specificity of the NS3-PKC interaction. The position of both fragments within the NS3 is schematically represented in Fig. 1. The assays performed were controlled with a further fragment of HCV polyprotein containing amino acid residues 1923-2043 (HCV polyprotein-(1923-2023)). All proteins were numbered according to their position within the HCV polyprotein (6).


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  		Fig. 1.   Schematic representation of the NS3 fragments used in this study. The top bar (open) represents the entire NS3 molecule. The shadowed bars below represent the expressed fragments of NS3: HCV polyprotein-(1189-1525) and HCV polyprotein-(1400-1615). The position of the arginine-rich sequence within NS3 is shown with a solid box. PThr represents the phosphorylatable threonine residue Thr1491 of the synthetic peptide that reproduces the arginine-rich domain of NS3 (12). The amino acid numbers were taken from Ref. 6.

Interaction between PKC and NS3-- We used the overlay method to analyze the specific physical interactions between NS3 or its derived peptide fragments and PKC. Equimolar amounts of the fusion proteins; GST-HCV polyprotein-(1189-1525), GST-HCV polyprotein-(1923-2043), and TRX-HCV polyprotein-(1400-1615) as well as of GST and TRX were immobilized on nitrocellulose sheets, and their binding to 125I-labeled PKC was determined. Both proteins GST-HCV polyprotein-(1189-1525) and TRX-HCV polyprotein-(1400-1615) were found to bind PKC. A lack of binding of the kinase to GST-HCV polyprotein-(1923-2043) as well as to GST and TRX demonstrated the specificity of these interactions. Interestingly, at all protein concentrations tested, GST-HCV polyprotein-(1189-1525) showed a higher binding capacity than TRX-HCV polyprotein-(1400-1615) (Fig. 2A).


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  		Fig. 2.   Binding of PKC to recombinant fragments of NS3 and inhibition of the binding by synthetic peptides that correspond to the arginine-rich domain of NS3 and to the autoregulatory domain of PKC. A, increasing amounts (0.1, 0.3, and 1 nmol) of GST-HCV polyprotein-(1189-1525), TRX-HCV polyprotein-(1400-1615), GST-HCV polyprotein-(1923-2023), GST, and TRX proteins were immobilized on nitrocellulose and overlaid with 125I-labeled PKC (50 nM). The nitrocellulose was washed, dried, and exposed to Kodak film for 4 h as described under "Experimental Procedures." B, increasing amounts of immobilized GST-HCV polyprotein-(1189-1525) and TRX-HCV polyprotein-(1400-1615) were overlaid with 125I-labeled PKC as described above in presence of a 5-fold molar excess of the peptides HCV (1487-1500) or PKC(19-36) in the overlaying buffer. The nitrocellulose was washed, dried, and autoradiographed for 4 h.

In further experiments, we analyzed the PKC binding to NS3 in competition binding assays. Increasing amounts of GST-HCV polyprotein-(1189-1525) were immobilized on nitrocellulose and incubated with 125I-labeled PKC in the presence of HCV polyprotein-(1487-1500) or PKC-derived peptide (PKC (19-36)) that reproduces the autoregulatory domain of the kinase (25). As shown in Fig. 2B, incubation with HCV (1487-1500) peptide reduced PKC binding to GST-HCV polyprotein-(1189-1525). The addition of PKC (19-36) peptide inhibited the binding to a similar extent. We performed a further study in which derivatives of HCV (1487-1500); a peptide with a scrambled sequence 1487-1500 and substituted analogues [Lys1487, Lys1488]HCV (1487-1500) and [Ile1487, Ile1488]HCV (1487-1500) were tested on their ability to inhibit the binding of the PKC to the immobilized GST-HCV polyprotein-(1189-1525). These compounds were previously demonstrated to bind and inhibit the PKC at a lower level compared with the parent peptide (12). The incubation of the immobilized GST-HCV polyprotein-(1189-1525) with 125I-labeled PKC in the presence of the derivatives of HCV (1487-1500) reveals, in comparison with the HCV (1487-1500) a reduced binding of PKC. These results suggest that the catalytic site of PKC binds NS3 via the arginine-rich region HCV (1487-1500).

It is conceivable that such an involvement of the catalytic site of the kinase in protein-protein interactions affects its activity. Indeed, our previous studies indicated that a similar interaction between PKA and NS3 resulted in an inhibition of PKA kinase activity (5). We therefore analyzed the substrate phosphorylation by PKC in the presence of HCV proteins in vitro. Experiments performed with histone IIIS as a substrate revealed a concentration-dependent inhibition of the kinase activity by GST-HCV polyprotein-(1189-1525). When myelin basic protein and histone H2B were used as substrates, the GST-HCV polyprotein-(1189-1525) concentrations that were required to give a 50% inhibition of substrate phosphorylation (IC50) varied significantly (Fig. 3A). The kinetic analysis of the inhibition revealed a mixed type of inhibition (Fig. 3B). Studies performed with TRX-HCV polyprotein-(1400-1615) also revealed significant differences between the IC50 for the phosphorylation of different substrates and a mixed type of inhibition. This suggests, in analogy to the inhibition of PKA by NS3 observed previously, a complex interaction between PKC, its substrate, and the inhibiting viral protein.


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  		Fig. 3.   Inhibition of the PKC-mediated phosphorylation by GST-HCV polyprotein-(1189-1525) and a plot demonstrating the mixed type of PKC inhibition relative to histone IIIS. A, the phosphorylation of protein substrates by PKC was performed in the presence of increasing concentrations of GST-HCV polyprotein-(1189-1525) as inhibitor. The substrates: histone IIIS (open circles), histone H2B (filled circles), and myelin basic protein (triangles) were phosphorylated at concentrations corresponding to their Km values (4.5 µM for histone IIIS, 2.5 µM for histone H2B, and 25 µM for myelin basic protein). The kinase activity toward each substrate in the absence of the inhibitor was referred to as 100%. The 32P incorporation into substrates was determined as described under "Experimental Procedures." B, PKC activity in the presence of GST-HCV polyprotein-(1189-1525) as inhibitor was investigated with histone IIIS. The substrate was phosphorylated at concentrations of 13.5, 4.5, and 1.5 µM corresponding to 3-, 1-, or <FR><NU>1</NU><DE>3</DE></FR>-fold of the Km of histone phosphorylation (filled triangles, open triangles, and squares, respectively). The PKC activity was determined as described above, and the data obtained were plotted according to Cornish-Bowden (49). The results shown are representative for three independent experiments.

Intracellular Distribution of Introduced HCV polyprotein-(1189-1525) and Peptide HCV (1487-1500)-- Previous studies that investigated the subcellular distribution of NS3 in HCV-infected cells demonstrated its cytoplasmatic localization (26). Moreover, NS3 fragments GST-HCV polyprotein-(1189-1525) and TRX-HCV polyprotein-(1400-1615) introduced into HEp-2 cells were also detected in the cytoplasm and in the perinuclear region (Ref. 8 and data not shown). In the following experiments, we examined the subcellular localization of the introduced GST-HCV polyprotein-(1189-1525) in neutrophils as well. The HCV protein was labeled with FITC and introduced into the cells, and the nuclei of the introduced cells were stained with Hoechst dye (33258). The results of the double labeling (Fig. 4) confirmed the subcellular localization of the introduced GST-HCV polyprotein-(1189-1525) and the exclusion of the protein from the nuclei. Immunoblot analysis of subcellular fractions obtained from GST-HCV polyprotein-(1189-1525)-introduced cells revealed that approximately 40% of the introduced protein was associated with the particulate and approximately 60% with the cytosolic fraction. Separation of cell organelles according to the method of Storrie and Madden (27) did not reveal any association of the introduced protein with cell organelles. These procedures affected the cell viability only marginally, as tested with trypan blue. The evaluated half-life of the introduced GST-HCV polyprotein-(1189-1525) was longer than 12 h (data not shown).


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  		Fig. 4.   Direct fluorescence microscopy to detect the cellular distribution of introduced GST-HCV polyprotein-(1189-1525). Fluorescein-derivatized GST-HCV polyprotein-(1189-1525) was introduced into neutrophils as described under "Experimental Procedures." The introduced cells were suspended in RPMI 1640 medium for 4 h before fixation. The slide was counterstained with Hoechst Blue (33258), and photographs were taken using Kodak 400 film with a 4-s exposure. A, FITC labeling. B, Hoechst Blue (33258) labeling.

Effects of NS3 on the Subcellular PKC Distribution-- Because NS3 binds and inhibits PKC, we investigated these interactions with regard to their ability to inhibit the translocation of PKC from the cytosolic to the particulate fraction. GST-HCV polyprotein-(1189-1525) as well as GST were introduced into the cells, and the effect of the proteins on the subcellular distribution of PKC was evaluated after treatment of the cells with 100 nM TPA for 5 min. Subsequently, the cytosolic and particulate fractions were separated and examined for their PKC content by immunoblot. The result indicates that the NS3 fragment was able to inhibit the TPA-induced redistribution of PKC (Fig. 5A). Very similar results were obtained with HEp-2 cells (results not shown). In contrast the treatment of the cells with HCV (1487-1500) peptide that strongly binds to PKC in vitro does not affect the extent of the TPA-induced cytosol-to-membrane translocation of the kinase. We therefore determined the distribution of the peptide and its interaction with PKC in the cell. The HCV (1487-1500) peptide was labeled with 3H introduced into neutrophils, and its half-life was determined as described under "Experimental Procedures." After 12 h approximately 80% of the introduced peptide was found in the cells. Subcellular fractionation followed by measuring the 3H activity revealed that approximately 97% of the labeled peptide was located in the cytosolic compartment and approximately 2-3% in the nuclear fraction. Only <0.5% of the peptide introduced was associated with the membrane fraction. The [3H]HCV (1487-1500)-introduced cells were then incubated with TPA for 5, 10, 20, or 30 min. Thereafter, the cytosolic and the particulate fractions were separated, and 3H radioactivity was measured as described. The results (Fig. 5B) demonstrate that TPA treatment induced a rapid translocation and a transient membrane association of [3H]HCV (1487-1500). These events were accompanied by a corresponding increase in immunoreactive PKC in the particulate fraction, when analyzed by counting the 125I-labeled bands obtained in an immunoblot with anti-PKC antibody (Fig. 5B).


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  		Fig. 5.   Effect of introduced HCV (1487-1500) peptide and GST-HCV polyprotein-(1189-1525) on the TPA-induced subcellular redistribution of PKC. Membrane association of HCV (1487-1500) peptide in course of TPA-induced PKC translocation. A, HCV (1487-1500) peptide, GST-HCV polyprotein-(1189-1525), and GST protein were introduced into neutrophils, and the cells were suspended in RPMI 1640 medium for 4 h as described under "Experimental Procedures." Thereafter, the introduced as well as control (not introduced) cells were treated with the carrier (Me2SO) or with 100 nM TPA for 5 min. Aliquots of a constant amount of the cells were lysed, and extracts from the cytosolic and the particulate fractions were applied to SDS/PAGE. The proteins were transferred onto nitrocellulose and subjected to immunoblot with an anti-PKC antibody. Immunoreactive bands were detected after treatment with a rabbit anti-mouse antibody followed by incubation with [125I]protein A. The blots were exposed for 14 h. Arrows indicate the positions of PKC. B, the labeled peptide [3H]HCV (1487-1500) was introduced into neutrophils as described above. The cells were treated with 100 nM TPA for 0, 5, 10, 20, or 30 min and lysed. Aliquots of the extracts from the particulate fraction were removed, and the 3H radioactivity was counted in a liquid scintillation counter. The remaining parts of the extracts were subjected to immunoblot with an anti-PKC antibody as described in A. The 125I-labeled PKC bands were cut out, and the radioactivity was counted in a liquid scintillation counter. The 3H radioactivity (open circles) and the 125I radioactivity of PKC band (closed circles) were measured in the particulate fraction. The results shown are representative for three independent experiments.

NS3 Inhibits TPA-induced Phosphorylation of p80-- Because TPA treatment activates PKC and induces the rapid phosphorylation of p80 in numerous cell systems (28), we compared the induction of cellular p80 phosphorylation by TPA in neutrophils harboring the introduced HCV protein. Cells were labeled with 32P for 2 h and then treated with TPA for 5 and 20 min. SDS/PAGE analysis of heat stable proteins extracted from the cells demonstrated that TPA-induced a rapid increase in the phosphorylation level of p80 and several other proteins in GST-HCV polyprotein-(1923-2043)-introduced neutrophils as well as in control (not introduced) neutrophils. A similar pattern of p80 phosphorylation was observed in GST-introduced cells. GST-HCV polyprotein-(1189-1525) reduced the TPA-induced increase in the phosphorylation level of the cellular heat stable proteins (Fig. 6A).


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  		Fig. 6.   Inhibition of the TPA-induced phosphorylation of p80 by GST-HCV polyprotein-(1189-1525), HCV (1487-1500), and HCV (1487-1500)-derived peptides in vivo and in vitro. A, GST-HCV polyprotein-(1189-1525) and GST protein were introduced into neutrophils as described above. The cells were metabolically labeled with 32P and during this labeling a treatment with 100 nM TPA for 0, 5, or 20 min was carried out. Heat stable proteins were extracted and separated by SDS/PAGE. The dried gel was exposed for 12 h. The migration of molecular mass protein standards is indicated at the left. B, the phosphorylation by PKC of heat stable proteins extracted from neutrophils in vitro was performed in the presence of increasing concentrations of the peptides HCV (1487-1500), [Ala1491]HCV (1487-1500), and [Val1491]HCV (1487-1500) as indicated. The phosphoproteins were separated by SDS/PAGE, and dried gels were exposed for 12 h. For the determination of the IC50, the 32P-labeled p80 bands were cut out, and the radioactivity was counted. C, peptides HCV (1487-1500), [Ala1491]HCV (1487-1500), and [Val1491]HCV (1487-1500) were introduced into the neutrophils, and the cells were labeled with 32P and treated with TPA as described for A. Heat stable proteins were extracted and the phosphorylation of p80 was analyzed as described above. Arrows indicate the positions of p80.

Surprisingly, no inhibition of TPA-induced p80 phosphorylation in vivo was seen in HCV (1487-1500) peptide-introduced neutrophils (Fig. 6C). A similar failure to inhibit the p80 phosphorylation by HCV (1487-1500) was also observed in other TPA-treated cells, i.e. HEp-2 and 3T3 cells (data not shown). Therefore, an experiment was performed to test the capacity of HCV (1487-1500) to inhibit PKC phosphorylation of p80 in vitro. The heat stable fraction of cellular proteins was prepared from neutrophils and phosphorylated by rat brain PKC in the presence of increasing concentrations of HCV (1487-1500). As shown in Fig. 6B, the phosphorylation of p80 in vitro was not significantly affected by the peptide at concentrations up to 50 µM. We have synthesized two peptides, analogous to HCV (1487-1500), in which the Thr1491 was replaced by alanine or valine. When the substituted derivatives were used for inhibition studies, the analogue [Ala1491]HCV (1487-1500) was found to be a potent inhibitor of the p80 phosphorylation (IC50 = 1.3 µM). Interestingly, the phosphorylation of several other heat stable proteins (of approximately 70 and 62 kDa) was only moderately affected by the peptide (Fig. 6B). No significant inhibition was observed with [Val1491]HCV (1487-1500) (IC50 > 50 µM). In subsequent experiments we compared these two peptides with regard to their ability to inhibit the p80 phosphorylation in vivo. The [Ala1491]HCV (1487-1500)- and [Val1491]HCV (1487-1500)-introduced cells were labeled with 32P and then treated with TPA for 5 and 20 min as described above. SDS/PAGE analysis of cellular heat stable proteins demonstrated that [Ala1491]HCV (1487-1500), but not [Val1491]HCV (1487-1500), reduced the TPA-induced phosphorylation of p80 (Fig. 6C).

NS3 Fragments Inhibit the Respiratory Burst of Neutrophils-- To ascertain the specificity of the interaction between NS3 and PKC, we examined the ability of the NS3 fragment to inhibit a further PKC-mediated process: the TPA-stimulated superoxide burst of neutrophils. The superoxide burst activity was measured in control (not introduced) neutrophils and in the cells that harbored the HCV proteins and HCV (1487-1500) peptide, as well as GST protein. Further experiments were performed with neutrophils treated with the known PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (29) and sangivamycin (30). As shown in Fig. 7, TPA-stimulated superoxide burst activity was inhibited by GST-HCV polyprotein-(1189-1525) and HCV (1487-1500) peptide. The inhibiting effect was comparable with the inhibitory potential of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and sangivamycin applied at concentrations corresponding to the 5-fold of IC50 determined with rat brain PKC.


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  		Fig. 7.   Inhibition of the TPA-induced respiratory burst of neutrophils by GST-HCV polyprotein-(1189-1525) and HCV (1487-1500) peptide. GST-HCV polyprotein-(1189-1525), GST-HCV polyprotein-(1923-2043), GST protein, and HCV (1487-1500) peptide were introduced into neutrophils. The cells were suspended in RMPI 1640 medium for 4 h as described above. The respiratory burst was measured by visible spectroscopy of superoxide dismutase-inhibitable reduction of cytochrome c. Data are normalized to the maximum rate of superoxide production by the control cells (not introduced neutrophils). Further control stimulations were performed on not introduced cells pretreated for 4 h with the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (150 µM) or sangivamycin (30 nM). The results shown are representative for five independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As the main result of this study we have shown that the enzymatic activity and the cytosol-to-membrane translocation of PKC is inhibited by the nonstructural protein 3 of hepatitis C virus. The arginine-rich sequence of NS3 Arg-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg, corresponding to amino acids 1487-1500 of HCV polyprotein and resembling the autoregulatory domain of PKC (25), is essential for this interaction. We have demonstrated that a synthetic peptide reproducing this sequence above acts as a PKC inhibitor in vitro and binds to and co-translocates with the kinase in vivo. When this sequence is embedded in a larger fragment of the NS3, it also binds to PKC and functions as an inhibitor of the kinase in vitro and in vivo. However, in contrast to the peptide, the larger recombinant NS3 fragment that contains this sequence prevents the translocation of PKC upon activation with TPA. These findings led us to conclude that PKC is immobilized by NS3 and thus does not translocate toward its intracellular membrane-associated receptor protein(s) RACK (31, 32). The model of the endogenous receptor protein for PKC suggests that the substrates and RACK may concomitantly bind the kinase (32). This is supported by the observation that the small molecule of the pepide HCV (1487-1500), which binds to the PKC catalytic site, does not inhibit the association of PKC with the particulate fraction. On the other hand, we have not ruled out the possibility that the RACK binding site(s) of PKC may also be involved in the binding to the NS3.

Although the pepide HCV (1487-1500) and the recombinant NS3 fragments bind PKC via its catalytic site, some differences in their interactions with the kinase were observed. The synthetic pepide HCV (1487-1500) serves as a substrate for the kinase and acts as a competitive inhibitor of PKC toward other substrates, whereas the NS3 fragments bearing the sequence are not phosphorylatable and inhibit the kinase in a mixed manner. On the basis of our previous report (8), we suppose that the arginine-rich sequence may only represent a part of the PKC-binding site on NS3 and that further regions of both molecules may participate in the binding. This view is supported by study that postulates a presence of polycation-binding sites on PKC. These sites are in strict positional relationships to the kinase active site (33). Thus the polycations may regulate the access of substrate to the active site (33, 34). Another possibility is that when the arginine-rich sequence is embedded in a larger fragment of NS3, other sites are exposed to the solvent and could interact with PKC (35). This could contribute to the explanation of why HCV polyprotein-(1189-1525) and nonphosphorylatable peptide [Ala1491]HCV (1487-1500) are better inhibitors of p80 phosphorylation than HCV (1487-1500). Finally, it is possible that some domains or fragments of NS3 determine the accessibility of the arginine-rich sequence as an anchoring site for PKC. Such a regulatory mechanism was previously demonstrated for the phosphorylation of full-length and truncated lipocortin and Ras-GAP (36, 37). The changes of the disposition of the arginine-rich sequence by other domains of NS3 could at least partially explain the different binding capacity of HCV polyprotein-(1189-1525) and HCV polyprotein-(1400-1615) toward PKC.

Our results have several important implications for the role of NS3 in the pathogenesis of the disease caused by HCV. First, the free shuttling of PKC between cellular compartments and the phosphorylation of target proteins, which are normal functions of the kinase, are down-regulated. Second, smaller fragments of NS3 containing the kinase binding sequence would be transported with the translocating enzyme. Because different PKC-isoforms translocate to distinct cellular compartments upon activation, the nuclear presence of biologically active helicase fragments (transported by the catalytic subunit of PKA (8) or beta -PKC (38)) is not unlikely. Similarly, wild-type p53 forms a complex with fragments of NS3 and transports them to the nucleus (39). Finally, according to the data presented here and in our previous works, the kinase inhibition mediated by the HCV (1487-1500) peptide or by NS3 fragments strongly depends on the nature of the substrate tested (5, 12). This observation predicts that NS3 or its fragments do not produce any uniform effect on the PKC-mediated signal transduction; rather pleiotropic effects on different signal pathways should be expected. The explanation of the interaction of NS3 with the intracellular signal pathways is further complicated by the fact that NS3 forms complexes with target proteins of protein kinases. The binding and formation of complexes with the substrates modulate the phosphorylation reaction. Such modulating effect of HCV and of related dengue virus NS3 was demonstrated in previous studies (5, 40).

The impairment of signal pathways through a direct binding of cellular signal proteins to viral antigens is not without a precedent. Numerous viral oncoproteins such as the simianvirus 40 large tumor antigen (41), the adenovirus E1B protein (42), or X protein of hepatitis B virus (43) form a complex with p53 and inactivate its functions. The bovine papillomavirus type 1 E5 oncoprotein binds to the platelet-derived growth factor recepor, mimics its dimerization, and activates its tyrosine kinase activity (44). Recently, such a direct protein-protein interaction was also demonstrated for the core protein of HCV (45). On the other hand, there are numerous examples of cellular proteins that are specific intracellular receptors for different protein kinases (46-48). In the case of an infection of a cell, the activity of the kinases may be a function of both the presence and the concentration of the receptors in the cell. The appearance of viral proteins that have anchoring site(s) for the kinase(s) may lead to a disturbance of the normal signal transduction. The mechanisms by which the HCV gene products exert their pathogenic effects are not known. Therefore, further studies of the mechanisms that act supplementary to the one described previously are required to determine the exact role of a given antigen in the cell.

    FOOTNOTES

* This work was supported in part by Deutsche Forschungsgemeinschaft Grant We 983/4-1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the 75th anniversary of Prof. Dr. H. Hilz, University of Hamburg.

§ To whom correspondence should be addressed. Tel.: 49-40-42818458; Fax: 49-40-42818378; E-mail: borowski@bni.uni-hamburg.de.

parallel Supported in part by grants from the Fritz-Ter-Meer Studienstiftung, Leverkusen.

    ABBREVIATIONS

The abbreviations used are: HCV, hepatitis C virus; NS3, nonstructural protein 3; PKA, protein kinase A; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; TPA, tetradecanoylphorbol-13-acetate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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