Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor–induced signal transduction by cleavage of the T-cell protein tyrosine phosphatase

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

The hepatitis C virus (HCV) is a worldwide major cause of chronic liver disease with a high tendency to establish a persistent infection. To permit persistent replication of viral genomes through the cellular translation machinery without affecting host cell viability, viruses must have developed mechanisms to control cellular cascades required for sufficient viral replication, on the one hand, and to adapt viral replication to the cellular requirements on the other hand. The present study aimed to further elucidate mechanisms by which HCV targets growth factor signaling of the host cell and their implications for viral replication. The study describes a novel mechanism by which HCV influences the activation of the epithelial growth factor receptor/Akt pathway through a nonstructural (NS)3/4A-dependent down-regulation of the ubiquitously expressed tyrosine phosphatase T cell protein tyrosine phosphatase (TC-PTP). NS3/4A is demonstrated to cleave TC-PTP protease-dependently in vitro at two cleavage sites. The in vivo relevance of this finding is supported by the fact that down-regulation of TC-PTP protein expression could also be demonstrated in HCV-infected individuals and in transgenic mice with intrahepatic expression of NS3/4A. Conclusion: This down-regulation of TC-PTP results in an enhancement of epithelial growth factor (EGF)-induced signal transduction and increases basal activity of Akt, which is demonstrated to be essential for the maintenance of sufficient viral replication. Hence, therapeutic targeting of NS3/4A may not only disturb viral replication by blocking the processing of the viral polyprotein but also exerts unforeseen indirect antiviral effects, further diminishing viral replication. (HEPATOLOGY 2009;49:1810–1820.)

Hepatitis C virus (HCV) is a major cause of chronic liver disease and hepatocellular carcinoma. Over 70% of HCV infections become chronic for reasons not well understood. The establishment of a persistent viral infection presupposes that the virus has developed strategies to subvert host antiviral defense and to use host cell infrastructure for its own replication in a directed way. Hence, a complex balance must exist to permit persistent replication of viral genomes and production of infectious particles and to simultaneously maintain host cell viability.

HCV has a 9.4 kb linear, single-stranded, positive-sense RNA genome encoding a polyprotein which is posttranslationally processed. Processing of the nonstructural part of the polyprotein is mediated by the nonstructural (NS)3/4A protease and helicase, which therefore is an intensely studied target for new antiviral drugs.1 Several HCV proteins appear to interfere with host antiviral response and other functions of the host cell, thereby promoting chronicity. Particularly, NS3/4A blocks the cellular interferon (IFN) response to double-stranded RNA by disruption of retinoic acid-inducible gene-I (RIG-I) signaling by proteolytic cleavage of CARD adaptor inducing IFNβ (CARDIF) and of Toll-like receptor (TLR)3 signaling by proteolytic cleavage of the adaptor molecule Toll/IL-1 receptor domain-containing adaptor inducing IFNβ (TRIF) (reviewed in Bode et al.2). NS3/4A has been further shown to modulate the intrahepatic immunity through reduction of type I/II dendritic cell and CD4+ T cell subsets, which is associated with an impaired inflammatory response toward hepatotoxic challenges such as lipopolysaccharide (LPS)/D-GalN or tumor necrosis factor (TNF)α/D-GalN.3

The knowledge of host proteins and signaling cascades essential for viral replication and the capacity of HCV to control the respective signaling pathways is fragmentary. Host factors that have been demonstrated to be essential for HCV replication are cyclophilin B, FK506-binding protein 8, the heat shock protein 90, and the so-called vesicle-associated membrane protein-associated proteins A and B.4 Moreover, studies indicate that HCV activates mammalian target of rapamycin (mTOR) and its substrate p70/S6 kinase through activation of the PI3K/Akt pathway,5–8 thereby increasing the survival of HCV-infected cells and suppressing viral replication.6, 7

The present report describes a novel pathway by which HCV influences activation of epithelial growth factor receptor (EGFR), which is highly expressed in the adult liver and plays an important role during liver development and regeneration.9

Abbreviations

CARDIF, CARD adaptor inducing IFNβ; EGF, epithelial growth factor; EGFR, epithelial growth factor receptor; HCV, hepatitis C virus; IFN, interferon; LPS, lipopolysaccharide; mTOR, mammalian target of rapamycin; NS, nonstructural; PI3K, phosphatidylinositol-3-kinase; PLCγ, phospholipase C γ; PP2A, protein phosphatase 2A; PTP, protein tyrosine phosphatase; RIG-I, retinoic acid-inducible gene-I; SHP2, src-homology phosphatase 2; TC-PTP, T cell protein tyrosine phosphatase; TLR, Toll-like receptor; TNF, tumor necrosis factor; TRIF, Toll/IL-1 receptor domain-containing adaptor inducing IFNβ.

Materials and Methods

Materials.

Restriction enzymes were from New England Biolabs (Frankfurt, Germany); Taq polymerase and recombinant hnEGF and hnIL-6 were from Roche (Mannheim, Germany), dNTP mix from Eppendorf (Hamburg, Germany), T4 DNA ligase from Promega (Madison, WI); oligonucleotides and small interfering (si)RNA were from MWG-Biotech (Ebersberg, Germany). Dulbecco's Modified Eagle Medium (DMEM)/nutrient mix F-12 was from Invitrogen (Karlsruhe, Germany) and fetal calf serum (FCS) from Perbio (Bonn, Germany). The major part, particularly of the phosphospecific antibodies, was from Cell Signaling Technology (Beverly, MA). Antibodies against EGFR and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), against AktP-Tyr326, β-actin, and HCV NS3 from Abcam (Cambridge, UK), against GAPDH from Biodesign (Saco, ME), against PP2A from Upstate (Charlottesville, VA), against PP2AP-Tyr307 from Epitomics (Burlingame, CA), against SrcP-Tyr418 and SrcP-Tyr529 from Calbiochem (Schwalbach, Germany), and against T cell protein tyrosine phosphatase (TC-PTP) from R&D Systems (Minneapolis, MN). Triciribine, AG1478, and LY294002 were from Calbiochem.

Cell Culture.

The human hepatoma cells HepG2 as well as the Huh7wt and the Huh9-13 cell line harboring the HCV replicase complex10 were cultivated in DMEM/nutrient mix F-12 supplemented with 10% (vol/vol) heat-inactivated FCS to a confluence of 80%. Medium was changed 16 hours before experiments were performed.

Samples from Murine Livers.

Livers from male 6 to 12-week-old mice with liver-specific expression of NS3/4A and from the corresponding wt mice (C57BL/6xCBA F1)3 were perfused with phosphate-buffered saline (PBS, pH 7.4, 4°C) until the livers turned pale. Thereafter, 100 to 125 mg liver tissue was taken for preparation of total protein lysates. The organ samples were incubated in 1 mL of buffer (see Supporting Information), homogenized, sonicated twice for 20 seconds at 4°C, and then used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Samples from Human Livers.

Liver tissue was either obtained from patients undergoing hemihepatectomy or lobectomy for removal of hepatocellular carcinoma or of liver metastasis or from patients undergoing biopsy for staging of liver disease after informed consent and in accordance with the guidelines of the local Ethics Committee of the University of Düsseldorf and the Declaration of Helsinki. Liver samples (15 cm3) were taken from tumor-free tissue, snap-frozen in liquid nitrogen, and stored at −70°C. Samples 1 and 2 of the control group were from adult organ donors whose livers were not completely used for transplantation and were kindly provided by Professor Martin Burdelski (University of Kiel). The preparation of protein lysates was performed as outlined for the murine samples.

Plasmids.

Standard cloning procedures were performed as outlined by Sambrook and Russel.11 All constructs were controlled by sequencing (MWG; Ebersberg, Germany). The cDNA for human TC-PTP was amplified from complementary (c)DNA generated from messenger (m)RNA of Huh7 cells by reverse transcriptase reaction and subcloned into the p3XFlag-CMV-7.1 expression vector (Sigma, Munich, Germany). The Flag-TC-PTP mutants with point mutations introduced into the two potential cleavage sites located at position amino acid (aa) 123/124 and aa 216/217 were generated by polymerase chain reaction (PCR) as outlined in the Supporting Information.

Real-time PCR.

Real-time (rt)PCR was performed as described.12 The primers used are listed in the Supporting Information. Specificity of rtPCR was controlled by no template and no reverse-transcriptase controls. Semiquantitative PCR results were obtained using the comparative threshold cycle (ΔCT) method and threshold values were normalized to hnSDHA.

Transfection Procedure and siRNA.

Huh cells were transiently transfected using Lipofectamine 2000 (Supporting Information and Ref.12). After transfection, cell culture was continued for a further 48 hours. Thereafter, experiments were performed as outlined in the Results section. siRNA (sequences are listed in Supporting Information) was transfected using the same protocol by adding in total 25 nM of the respective siRNAs.

Generation of HepG2 NS3/4A/Mock Cells.

The pCFG5-IEGZ retroviral vector allowing expression of green fluorescent protein (GFP) of the same mRNA as the gene of interest was used to generate NS3/4A cDNA containing retrovirus (giving HepG2-NS3/4A) or control virus (giving HepG2-mock) for transfection of HepG2 cells as outlined13 and described in the Supporting Information.

Western Blot Analysis.

At the end of experimental treatment, cells were washed twice with PBS supplemented with 0.1 mM Na3VO4, solubilized in lysis buffer, and sonicated 2× for 20 seconds at 4°C. Protein concentration was estimated by using the BioRad protein assay. Equal amounts of protein were subjected to Western blot analysis (Supporting Information and12.

In Vitro TC-PTP Cleavage.

The in vitro assay for cleavage of TC-PTP by NS3/4A was performed using lysates from Huh7 cells transiently transfected with the p3XFlag-hnTC-PTP expression vector encoding wildtype Flag-TC-PTP or Flag-TC-PTP where the potential NS3/4A-cleavage sites at position aa 123/124 and aa 216/217 have been mutated (Supporting Information). Cells were lysed 48 hours after transfection using 100 μL of lysis buffer; 30 μg of protein were adjusted to 60 μL with protease assay buffer and started by adding recombinant HCV NS3/4A protease or HCV NS3/4A protease-dead mutant obtained from Anaspec (San Jose, CA) at the concentrations indicated in the figure legends. Samples were incubated at 37°C for 3 hours. The reaction was stopped by adding 20 μL of Laemmli buffer and denaturation at 95°C for 5 minutes.

Statistical Evaluation.

The densitometric evaluation of the protein bands obtained by Western blot analysis was processed with the Molecular Imaging Software (v. 4.0.3) from Kodak. The ratio of the net intensity of the phosphorylated protein band and the band of the corresponding unphosphorylated protein was calculated. The ratio representing the untreated control was set to 1 and data are displayed as a percentage of the control. Standard deviations for controls were calculated by referring all controls to the mean, which was set to 1. The root mean square deviation is indicated. The significance (P < 0.05) was calculated as an unpaired Student's t test.

Results

HCV Sensitizes Cells for EGF Signaling.

As shown in Fig. 1, EGF-induced phosphorylation of EGFR at the tyrosine residues Tyr845, Tyr992, Tyr1068, and Tyr1173 but not at Tyr1045 was up-regulated up to three-fold of control conditions in the presence of the HCV replicase complex (Huh9-13). Likewise, EGF-mediated activation of phospholipase C γ (PLCγ) was increased in Huh9-13 cells (Fig. 2A), whereas the activating phosphorylation of Akt at Thr308 and even stronger at Ser473 was already strongly enhanced under basal conditions (Fig. 2B,C, quantified in Fig. 2D) and was further increased upon stimulation with EGF (Fig. 2B). Inhibition of EGFR activation by AG1478 completely abrogates the EGF-induced tyrosine phosphorylation of EGFR at Tyr845 and Tyr1068 as well the EGF effects on Akt phosphorylation at Ser473 (Fig. 2E). However, the increased basal activity of Akt observed in HCV replicon expressing cells was not affected by EGFR inhibition, indicating that this is independent from EGFR activity.

Figure 1.

HCV sensitizes cells for EGF. (A) Huh9-13 cells and Huh7 cells were stimulated with 100 ng/mL EGF or 200 U/mL IL-6 as indicated. Thereafter total protein extracts were prepared according to Materials and Methods and 30 μg of protein were analyzed by immunoblot for tyrosine phosphorylation of EGFR at Tyr845, Tyr992, Tyr1045, Tyr1068, and Tyr1173 and of STAT3 at Tyr705 using phosphospecific antibodies. Total EGFR and STAT3 expression was analyzed using the respective antibodies. (B) Densitometric analysis of the EGFR phosphorylation after 20 minutes of EGF stimulation was performed. Phosphorylation of the respective tyrosine residue was normalized to the values determined for the total EGFR protein. The results are expressed relative to the respective control, which was set to 1. Data are presented as means ± standard deviation (n = 3) and significant differences from control conditions are indicated with asterisks (*).

Figure 2.

HCV enhances basal and EGF-induced activation of Akt. (A) Huh9-13 and Huh7 cells were stimulated with 100 ng/mL EGF or 200 U/mL IL-6, whereas (B) the depicted concentrations of EGF were used. After the times indicated total protein extracts were prepared. (C) Protein extracts of untreated Huh7 and Huh9-13 cells were used, and (E) cells were pretreated with 10 μM AG1478 or DMSO for control prior to stimulation with EGF (100 ng/mL) as indicated. Thirty μg of total protein extracts were analyzed by immunoblot using antibodies specific for tyrosine-phosphorylated PLCγ1Tyr771 for (A) and phosphospecific antibodies specifically recognizing phosphorylated AktSer473 (B,C,E), phosphorylated AktThr308 (C), and EGFR phosphorylated at Tyr845 and Tyr1068 (E). Total protein levels of PLCγ1, Akt, NS3, and GAPDH were determined using specific antibodies as depicted. (D) Densitometric analysis and quantification of basal Akt-phosphorylation at Ser473 and Thr308 in Huh7 and Huh9-13 cells was performed as outlined in Materials and Methods and the legend to Fig. 1. Data are presented as means ± standard deviation (n = 4). Significant differences are indicated with asterisks (*).

NS3/4A Mediates Down-Regulation of TC-PTP.

Enhanced EGFR phosphorylation at the different tyrosine residues could be due to decreased phosphatase-dependent dephosphorylation. Interestingly, the protein levels of TC-PTP (Fig. 3A), but not of SHP2 or PTP1B (Supporting Information), which are all known negative regulators of EGFR,14–16 were strongly reduced in Huh9-13 cells when compared to control cells or to Huh9-13 cells cured from the replicon by treatment with IFNα (Fig. 3A). This effect of HCV is not due to down-regulation of TC-PTP on the level of mRNA expression, as indicated from the analysis of the TC-PTP mRNA levels using rtPCR (Supporting Information). Inhibitor studies using MG132, bafilomycin A, and the pan-caspase inhibitor Z-VAD-FMK further indicate that HCV-mediated down-regulation of TC-PTP protein levels neither involves proteasomal degradation nor lysosomal or caspase-mediated proteolysis (Supporting Information).

Figure 3.

The HCV NS3/4A protease down-regulates TC-PTP, enhances basal activation of Akt, and sensitizes cells for EGF. (A) Huh9-13 cells were cleared from the subgenomic HCV replicon by repeated cycles of IFNα treatment and compared with untreated Huh9-13 cells or with respective control cells (Huh7). Cells were stimulated with EGF for 20 minutes or left untreated as indicated. (B) HepG2 cells stably expressing NS3/4A or respective control cell lines were stimulated with different concentrations of EGF as indicated. Total protein extracts were prepared and 30 μg of protein were analyzed by immunoblot using (A) antibodies specific for TC-PTP, HCV NS3, or GAPDH and (B) antibodies specifically recognizing phosphorylated AktSer473 and PLCγ1Tyr771. Total TC-PTP, PLCγ1, Akt, HCV NS3, and GADPH protein levels were detected using the respective antibodies. (C) Total protein lysates from liver tissue of transgenic mice with liver-specific expression of NS3/4A (lanes 6-10) or from the corresponding wildtype mice (lanes 1-5) were prepared as outlined in Materials and Methods and 100 μg of protein were analyzed by immunoblot using antibodies specifically recognizing EGFR phosphorylated at Tyr845, Tyr992, Tyr1068, or Tyr1173 or AktSer473. In addition, blots were analyzed for expression of TC-PTP and GAPDH.

Down-regulation of TC-PTP at the level of protein (Fig. 3B) but not of mRNA (Supporting Information) could also be observed in HepG2 cells stably expressing NS3/4A when compared with mock transfected cells, indicating that NS3/4A alone is sufficient to mediate the reported effects of the HCV replicon. According to this, NS3/4A-dependent down-regulation of TC-PTP was accompanied by an enhancement (Fig. 3B) of basal Akt activity and of EGF-induced activation of Akt and PLCγ. In support of the data from cell culture, TC-PTP levels were strongly reduced in liver tissue from NS3/4A transgenic mice (Fig. 3C), whereas the EGFR phosphorylation at Tyr845, Tyr992, Tyr1068, and Tyr1173 as well as the activating phosphorylation of Akt at Ser473 was shown to be enhanced.

In vitro treatment of lysates from Flag-TC-PTP or control transfected Huh7 cells with different concentrations of purified recombinant NS3/4A resulted in decreased protein levels of exogenous Flag-TC-PTP as well as of endogenous TC-PTP (Fig. 4A) which was not observed if a protease dead-mutant of NS3/4A was employed, suggesting that TC-PTP is protease-dependently destructed by NS3/4A. Both the 45 kD and the 48 kD isoform of TC-PTP comprise two Cys-(Ser/Ala) peptide bonds at position aa 123/124 (Cys/Ala) and aa 216/217 (Cys/Ser), which represent potential cleavage sites for NS3/4A. Cleavage at position 216 would result in the release of an N-terminal 216 aa fragment (in the case of the Flag-tagged molecule 234 aa) and a C-terminal fragment of 199 aa. Cleavage of the N-terminal fragment at the second cleavage site at position 123/124 would result in two fragments of 123 aa (in the case of the Flag-tagged molecule 141 aa) and 93 aa size. As shown in Fig. 4A, treatment of Flag-TC-PTP expressing lysates with NS3/4A leads to the release of a fragment of ≈30 kD (Flag-F1) and a second of ≈20 kD (Flag-F2) size which are both recognized by the Flag and by the TC-PTP antibody and roughly correspond to the size expected for the two N-terminal fragments of 234 aa (Flag-F1) and 141 aa (Flag-F2) size. Apart from this reprobing of the blot with the TC-PTP specific antibody directed against the N-terminal part of TC-PTP leads to the detection of a third fragment (F′) of ≈25 kD size, which most likely represents the untagged N-terminal 216 aa fragment of the endogenous TC-PTP. According to this, mutation of the cleavage site located at position 123/124 led to the disappearance of the Flag-F2 fragment, whereas the Flag-F1 fragment becomes undetectable upon deletion of the site located at position 216/217.

Figure 4.

HCV replication or NS3/4A expression impair TC-PTP expression in vivo and the NS3/4A protease/helicase cleaves TC-PTP in vitro. (A) Huh7 cells were transfected with 2.5 μg of the Flag-TC-PTP expression vector or the respective vector encoding mutant Flag-TC-PTP with mutations in the different cleavage sites located at position aa 123/124 (Flag-TC-PTP123/124mut) or aa 216/217 (Flag-TC-PTP216/217mut). The protease assay was performed by adding the indicated amounts of the purified recombinant NS3/4A (wt) or the purified recombinant protease-dead mutant of NS3/4A (mt) to total protein lysates. After 3 hours the reaction was stopped and 40 μL of the protease assay reaction was analyzed for TC-PTP and GAPDH expression. (B) 30 μg of protein extracts derived from liver tissue of HCV-infected patients or respective controls (see Table 1) were analyzed for TC-PTP and β-actin expression by immunoblot. (C) Densitometric analysis of the results of (B) was performed and the results are expressed relative to control No. 1, which was set to 1. Data are presented as means ± standard error of the mean (control group n = 12 and HCV group n = 11) and significant difference is indicated with an asterisk (*).

Next, the analysis was extended from experimental models to HCV-infected patients. Corresponding to the findings reported above, TC-PTP was also strongly down-regulated in liver tissue from 11 HCV-infected patients when compared to respective controls (Fig. 4B,C; Table 1), indicating that TC-PTP protein expression is substantially impaired in the liver of patients with chronic, persisting HCV infection.

Table 1. Individuals from Whom Protein Extracts Derived from Liver Samples Were Analyzed for Levels of Protein as Specified in Fig. 4
No.GenderAgeDiagnosisHistologyALTBilirubinQuick
  1. Histology revealed no cirrhosis (0), minimal (I), mild (II), severe (III) fibrosis or cirrhosis (IV). HCV genotype is indicated in parentheses.

  2. Abbreviations: ALT, alanine aminotransferase; CA, cancer; C2, alcohol; CCC, cholangiocarcinoma; HBV, hepatitis B virus infection; HCC, hepatocellular carcinoma; LM, liver metastasis; NASH, nonalcoholic steatohepatitis; nd, not determined. Normal values for bilirubin <1.1 mg/dL, ALT <23 U/L, and Quick 70%-110%. For samples 1 and 2, see Materials and Methods.

3Female63NASH, HCCIV130.689
4Male64C2 abuse, HCCI150.484
5Female69HCCI400.5104
6Female29HBV, HCCII840.495
11Female68CCC01072.0174
12Female55CCCI350.5100
13Female63CCCI200.883
14Female65LM, Colon CA01140.978
15Male64LM, Colon CAI400.680
16Female65CCC0754.2360
7Male58HCV (1)IV1320.7107
8Female42HCV (1)0280.4101
9Female51HCV (1)0240.7101
10Male44HCV (1)II180.8104
17Male29HCV (nd)I1830.7104
18Male48HCV (1)II1200.5101
19Female47HCV (nd)I680.890
20Female67HCV (nd)I320.785
21Male66HCV (nd)0280.884
22Male69HCV (1), HCCIV552.0483
23Female63HCV (1), HCCIII590.962

Suppression of TC-PTP Up-Regulates EGF-Induced Phosphorylation of EGFR and Enhances Basal and EGF-Induced Akt Activity.

TC-PTP can counteract EGF-induced tyrosine phosphorylation of EGFR, thereby enhancing downstream signal-transduction through PI3K/Akt in Cos1 cells, HeLa cells, and mouse fibroblasts.16, 17 As shown in Fig. 5A, specific knockdown of TC-PTP in Huh7 cells leads to enhanced EGF-induced phosphorylation of the EGFR at Tyr845, Tyr992, Tyr1068, and Tyr1173 and results in a substantial increase of EGF-induced Akt activation, which corresponds to the observations in the presence of the HCV replicon. Hence, these data indicate that down-regulation of TC-PTP is indeed responsible for the effects of the HCV replicon on EGFR/Akt signaling. Moreover, although TC-PTP is a tyrosine phosphatase, the basal activating phosphorylation of Akt was strongly enhanced upon down-regulation of TC-PTP levels, indicating that TC-PTP indirectly also controls basal Akt activity. This control of basal Akt activity by TC-PTP does not occur through regulation of c-Src phosphorylation at Tyr418, which has been demonstrated to be a target of TC-PTP,18 nor phosphorylation of Akt at Tyr326, which is the phosphorylation site for c-Src-mediated phosphorylation,19 as indicated from experiments using Huh7 cells transfected with TC-PTP-specific siRNA. Likewise, the activating phosphorylation of the phosphatase PTEN, one of the major negative regulators of Akt signaling, at Ser380 and Thr382/383 remains unchanged in cells depleted of TC-PTP by specific siRNA (Fig. 5B). Another negative regulator of Akt is the serine/threonine phosphatase PP2A,20 which itself is thought to be negatively controlled by phosphorylation at Tyr307.21 However, although phosphorylation of PP2A at Tyr307 was enhanced upon down-regulation of TC-PTP by specific siRNA (Fig. 5B), phosphatase activity of PP2A remained unchanged (Supporting Information). Hence, these data indicate that the effect of TC-PTP on basal Akt activity does not depend on regulation of c-Src, PTEN, or PP2A.

Figure 5.

TC-PTP negatively regulates EGF-dependent signaling as well as basal Akt activity, which may involve deregulation of PP2A activity. (A) Huh7 cells were transfected with TC-PTP specific siRNA or with GFP siRNA for control. Cells were treated with EGF as indicated or left untreated for control. Analysis of protein lysates for phosphorylation of EGFR at Tyr845, Tyr922, Tyr1068, and Tyr1173 and of Akt at Ser473 and for expression of GAPDH, Akt, and EGFR was performed by immunoblot as outlined in the figure legends to Figs. 1 and 3. (B) Total protein extracts from unstimulated Huh7 cells transfected with TC-PTP siRNA or GFP siRNA were prepared 2 days after transfection and analyzed for phosphorylation of Akt at Ser473, Thr308, or Tyr326, of Src at Tyr418 or Tyr529, of PP2A at Tyr307, and of PTEN at Ser380, Thr382, and Thr383 using specific antibodies. Total protein levels of TC-PTP, Akt, Src, PP2A, PTEN, and GAPDH were determined using the respective antibodies. Densitometric analysis of the blots from (B) was performed as outlined in Materials and Methods and the legend to Fig. 1 and the results for those phosphorylation sites which were significantly regulated in the absence of TC-PTP are presented as graphs.

Akt Activity Is Critical for HCV Replication.

The data presented herein indicate that HCV enhances ligand-induced EGFR signaling and leads to constitutive activation of Akt, which results from NS3/4A-mediated suppression of TC-PTP. To further clarify the possible implications of these interactions for HCV replication the effect of inhibitors supposed to specifically suppress activation of EGFR (AG1478), of PI3K (LY294002), and of Akt (Triciribine) on replication of the HCV replicon was examined in the presence or absence of EGF. As shown in Fig. 6A,B, stimulation of Huh9-13 cells with EGF resulted in an enhanced abundance of HCV mRNA. This effect of EGF could be abrogated by inhibition of EGFR activity by AG1478 (Fig. 6A) or of PI3K activity by LY294002 (Fig. 6B) at concentrations of 1 and 10 μM, whereas basal HCV replication remained unchanged. In contrast, treatment of Huh9-13 cells with Triciribine, which is thought to specifically block Akt activity without affecting upstream activators of Akt,22 time- and dose-dependently affected both basal (Fig. 6C) as well as EGF-enhanced (Fig. 6D) replication. Correspondingly, suppression of Akt expression by specific siRNA resulted in a strong reduction of the abundance of viral mRNA in unstimulated and in EGF-stimulated (Fig. 6E) Huh9-13 cells. The inhibitory effect of Akt inhibition on HCV replication was not due to changes of cellular viability (Supporting Information), as indicated by using the WST1 assay. These data suggest that activation of EGFR enhances replication of HCV but is not essential for replication of HCV, whereas constitutive activation of Akt seems to also be important for efficient HCV replication in an EGFR-dependent and -independent manner.

Figure 6.

Inhibition of EGFR activation and of PI3K activity abrogates EGF-induced enhanced HCV replication, whereas inhibition of Akt affects basal replication. Huh9-13 cells were either treated with DMSO for control or with (A) 1 or 10 μM AG1478 for 24 hours, (B) 1 or 10μM LY294002 for 48 hours, (C) 5 or 20 μM Triciribine for the times indicated, and (D) 5 or 20 μM Triciribine for 48 hours in the absence or presence of 50 ng/mL EGF as indicated. (E) Huh9-13 cells were transfected with Akt-specific siRNA or GFP siRNA for control 48 hours prior to stimulation with EGF (50 ng/mL) as indicated. Total RNA was prepared and subjected to RT-PCR for NS3 mRNA abundance as outlined in Materials and Methods. Semiquantitative PCR results were obtained using the ΔCT method. NS3 mRNA was normalized to hnSDHA and relative NS3 mRNA levels were expressed as fractions of the normalized value of the control, which was set to 1. Data are presented as means ± standard deviation and significant differences are indicated with asterisks (*).

Discussion

The present study shows that HCV down-regulates the expression of TC-PTP in vivo and in vitro. This results in enhancement of EGF-induced signaling and increased basal Akt activity, which is critical for efficient replication of HCV. The molecular basis of TC-PTP down-regulation is most likely the proteolytic cleavage of TC-PTP by the NS3/4A protease/helicase of HCV, as indicated from in vitro data showing that wildtype NS3/4A but not a protease-dead mutant of NS3/4A cleaves TC-PTP. NS3/4A is a serine protease that catalyzes cleavage at Cys-(Ser/Ala) peptide bonds. The data presented herein indicate that NS3/4A cleaves TC-PTP at two Cys-(Ser/Ala) peptide bonds that are located at positions aa 123/124 (Cys/Ala) and aa 216/217 (Cys/Ser), because mutation at the respective site renders TC-PTP resistant against NS3/4A-mediated cleavage at these sites. The data from Western blot analysis of liver tissue from HCV-infected patients and from transgenic mice with liver-specific expression of NS3/4A suggests that this down-regulation of TC-PTP also occurs in vivo. Considering that in most studies not more than 10% of hepatocytes from infected liver tissue express HCV proteins at levels that are detectable for immunohistochemistry,23 the observed extent of down-regulation of TC-PTP is surprising and might indicate that, apart from cleavage, other, possibly indirect mechanisms are involved that are much more effective. However, it should be noted that CARDIF, another reported substrate of NS3/4A, has been demonstrated to be almost completely cleaved in at least two of four liver tissue samples from HCV-infected patients where only the residual cleavage product was detectable.24 This might indicate that, although not detectable for immunohistochemistry, the distribution and the activity of NS3/4A is sufficient enough to effectively cleave the respective target proteins in infected tissue. In line with this assumption a previous study using an in situ hybridization technique reported that a much higher percentage of hepatocytes than originally expected of up to 80% was found to be positive for both positive- and negative-strand HCV RNA.25

The reduction of TC-PTP protein levels results in an enhancement of ligand-induced activation of EGFR and downstream signaling toward Akt and PLCγ. These data are in line with previous reports demonstrating that TC-PTP dephosphorylates EGFR and the adaptor protein p52shc, thereby inhibiting downstream signaling through activation of the PI3K/Akt pathway and of c-Jun N-terminal kinase.16

Apart from enhancing EGFR signaling, suppression of TC-PTP either by siRNA or by NS3/4A also resulted in increased basal phosphorylation of Akt at Ser473 and Thr308, which occurred independently from activation of EGFR. This indicates that TC-PTP is not only an important negative regulator of EGF-induced signaling but also prevents Akt kinase from being ligand-independently activated. Akt activity is mainly governed by phospholipid-binding and PDK1-mediated phosphorylation of Thr308 and by phosphorylation within the carboxy terminus at Ser473.19 Because TC-PTP is a tyrosine phosphatase, control of basal Akt activity by TC-PTP must occur indirectly either through regulation of a serine/threonine phosphatase or of a serine/threonine kinase. The data presented herein indicate that this regulation of Akt activity by TC-PTP involves neither c-Src-mediated Akt phosphorylation at Tyr418 nor the serine/threonine phosphatases PTEN or PP2A, which are known to negatively regulate Akt activity. Thus, the characterization of the mechanism responsible for the enhanced basal Akt activity observed in the absence of TC-PTP remains to be established.

In line with the results of this study, previous reports demonstrated that HCV induces activation of the PI3K pathway mediating activation of Akt as well as of mTOR and the p70/S6 kinase downstream of mTOR.7, 8 This activation of PI3K/Akt signaling by HCV is thought to occur through induction of oxidative stress8 and an interaction of NS5A with the p85 subunit of the PI3 kinase, contributing to an increased survivability of NS5A-expressing cells.5, 26 Hence, considering the data presented herein, it is well conceivable that suppression of TC-PTP by NS3/4A disrupts the endogenous negative control of EGF- and Akt-mediated signaling, making these pathways more sensitive for activation by oxidative stress and/or the action of NS5A. The resulting enhancement of EGF signaling, and in particular of basal Akt activity, is demonstrated to be essential for sufficient replication of HCV. However, it remains to be clarified which of the multiple signaling events elicited by activation of Akt are required for these effects. With respect to this, previous reports indicate that at least Akt-mediated activation of the mTOR/p70S6 kinase cascade is not involved because activation of this pathway has been demonstrated to transmit repressive rather than enhancing effects on HCV replication.6, 7

Apart from this, the pathophysiological implications of HCV-induced activation of PI3K/Akt signaling could be manifold. Several reports have indicated that activation of the PI3K/Akt pathway is involved in the protection of infected cells from apoptosis, which might play a role in the pathogenesis of HCC26 and of HCV-associated B-cell lymphoma.27 Our study extends this knowledge by defining two responsible key factors that are necessary and sufficient for these effects and thus guide future therapeutic intervention strategies: the NS3/4A protease represents the viral culprit that targets the host protein TC-PTP. Therefore, therapeutic targeting of NS3/4A by protease inhibitors would not only block viral replication by inhibiting the processing of the viral polyprotein, but also deprives the virus of an important option to exploit host cell signaling to its own purposes.

Acknowledgements

We thank Dr. Ludwig (Department of Molecular Virology, University of Münster, Germany) for the ability to generate NS3/4A-expressing HepG2 cells and respective mock cell lines by retroviral gene transfer.

Ancillary