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. 2007 Jul 16;40(4):508–521. doi: 10.1111/j.1365-2184.2007.00453.x

Mitogen‐activated protein kinase signalling pathways triggered by the hepatitis C virus envelope protein E2: implications for the prevention of infection

L‐J Zhao 1, P Zhao 1, Q‐L Chen 1, H Ren 1, W Pan 1, Z‐T Qi 1
PMCID: PMC6496583  PMID: 17635518

Abstract

Abstract.  Objective: Hepatitis C virus (HCV) is a major pathogenic factor of liver diseases. During HCV infection, interaction of the envelope protein E2 of the virion, with target cells, is a crucial process for viral penetration into the cell and its propagation. We speculate that such interaction may trigger early signalling events required for HCV infection. Materials and methods: Human liver cell line L‐02 was treated with HCV E2. The kinase phosphorylation levels of mitogen‐activated protein kinase (MAPK) signalling pathways in the treated cells were analyzed by Western blotting. The proliferation of the E2‐treated cells was evaluated by MTT assay. Results: HCV E2 was shown to be an efficient activator for MAPK pathways. Levels of phosphorylation of upstream kinases Raf‐1 and MEK1/2 were seen to be elevated following E2 treatment and similarly, phosphorylation levels of downstream kinases MAPK/ERK and p38 MAPK also increased in response to E2 treatment, and specificity of kinase activation by E2 was confirmed. E2‐induced MAPK/ERK activation was inhibited by the MEK1/2 inhibitor U0126 in a concentration‐dependent manner. Blockage of relevant cellular receptors reduced activation of Raf‐1, MEK1/2, MAPK/ERK and p38 MAPK by E2, indicating efflux of the E2 signal from extracellular to the intracellular spaces. Thus, kinase cascades of MAPK pathways were continuously affected by E2 presence. Moreover, enhancement of cell proliferation by E2 appeared to be associated with the dynamic phosphorylation of MAPK/ERK and p38 MAPK. Conclusion: These results suggest that MAPK signalling pathways triggered by E2 may be a potential target for prevention of HCV infection.

INTRODUCTION

Infection with hepatitis C virus (HCV) is an important cause of many human liver disorders. Prevalence of HCV infection is approximately 1% in Europe and the United States, and an estimated 1% to 20% in China, Japan and Africa (Knoll et al. 2001). Conditions of individuals infected with HCV frequently progress to chronic hepatitis C, cirrhosis and hepatocellular carcinoma. Strategies for prevention and control of HCV infection could be improved based on the knowledge of HCV pathogenesis.

Both direct viral pathogenic effects and host immune responses contribute to human diseases associated with viruses. Here, the investigation is pursued to elucidate whether disturbance of cell signal transduction caused by viruses is involved in viral pathogenesis. Some signalling cascades are interfered with by a variety of viruses and such interference may account for viral pathogenicity. Modulation of transforming growth factor‐β 1‐mediated signalling pathway by the Epstein‐Barr virus is one possible molecular basis for viral oncogenicity (Fukuda et al. 2002). Signal transduction mediated by G protein‐coupled receptor of Kaposi's sarcoma‐associated herpesvirus is responsible for the regulation of host gene expression (Polson et al. 2002). Mitogen‐activated protein kinase (MAPK) pathways are often in focus due to their involvement in viral persistence and tumourigenesis. For instance, up‐regulation of the MAPK pathway by the papillomavirus E6 oncoprotein has been found to be involved in tumourigenesis (Chakrabarti et al. 2004), and the Ras/RalGEF/p38 signalling pathway plays an important role in regulation of reovirus replication and oncolysis (Norman et al. 2004). During primary infection, Kaposi's sarcoma‐associated herpesvirus manipulates host cells to facilitate viral entry and postentry productive lytic replication by modulating multiple MAPK pathways (Pan et al. 2006). Cell membrane accumulation of influenza A virus haemagglutinin triggers nuclear export of the viral genome via activation of the MAPK signalling cascade (Marjuki et al. 2006). These results indicate that viral pathogenicity might be in part attributed to interference by viruses of cellular signalling pathways. As for HCV, core protein and nonstructural proteins display properties implicated in HCV pathogenicity by affecting diverse signal molecules such as JAK1/2, STAT‐1, STAT‐3, NF‐κB and AP‐1 (Gong et al. 2001; Kim et al. 2001; Hosui et al. 2003; Macdonald et al. 2003; Bataller et al. 2004).

Hepatitis C virus has positive‐stranded RNA genome that encodes a polyprotein of about 3000 amino acid residues. The polyprotein is cleaved by viral and cellular proteases to generate 10 proteins, including core protein, envelope protein 1 (E1), E2, p7, nonstructural protein (NS) 2, NS3, NS4A, NS4B, NS5A and NS5B. E2 exposed on the virion mediates attachment of HCV to target cells through interaction with receptors. There are several cellular surface molecules identified as HCV receptors; human CD81 is a cell molecule involved in signal transduction (Levy et al. 1998), CD81, a putative HCV receptor, interacts with E2 protein that then influences cellular functions (Pileri et al. 1998; Cormier et al. 2004). Low‐density lipoprotein receptor (LDLR) is considered to be a coreceptor for HCV, as it mediates HCV entry into susceptible cells (Agnello et al. 1999). Receptor‐mediated signal transduction is an initial event where cells recognize a ligand and bind to it, followed by delivery of the extracellular signal to the intracellular signal network. HCV E2‐plasmamembrane receptor interaction is an essential process, not only for virion invasion into the cell, but also for the initial signalling event following HCV infection. It is thus necessary to fully investigate signalling pathways triggered by the HCV E2 protein and then the cellular consequences.

The MAPK pathways, composed of ERK, Jun kinase (JNK) and p38 MAPK subfamilies, are the focus of intensive research because of their importance in controlling many important cellular functions (Johnson & Lapadat 2002). The ERK pathway results in stimulation of cells to growth factors and is responsible for cell proliferation and differentiation. JNK and p38 MAPK pathways respond to stress stimuli and contribute to the inflammatory response and to apoptosis. Inhibition of MAPK pathways is a popular exploration for the control of human cancers and inflammatory diseases. Novel anticancer drugs aimed at targeting MAPK pathways are always under clinical investigation (Hilger et al. 2002). Here, we presume that HCV tropism for target cells may evoke alterations in MAPK pathways implicated in viral pathogenesis. We have reported previously that HCV E2 protein promoted human hepatoma Huh‐7 cell proliferation through activation of MAPK/ERK and up‐regulated MAPK/ERK and p38 MAPK in T‐lymphoma Molt‐4 cells (Zhao et al. 2005; Zhao et al. 2006). A number of cell lines, primary cultures, transient transfection systems and transgenic mice are valuable tools used to reveal signalling events. L‐02 is a human normal liver cell line and is used as a cell system for evaluation of responses to various compounds (Yu et al. 2000; Ji et al. 2002; Gao et al. 2003; Chen & Yan 2005). In the present study, L‐02 is chosen to be a cell model for investigation of the kinase cascades of MAPK pathways initiated by the HCV E2 protein. Results achieved with L‐02 cells are the latest developments following from our previous findings in hepatoma and lymphoma cells.

MATERIALS AND METHODS

Materials

Soluble purified HCV E2 protein (genotype 1a) derived from Chinese hamster ovary cells and the monoclonal antibody (mAb) against the E2 protein were generously provided by Dr Michael Houghton (Chiron Corporation, Emeryville, CA, USA). Goat anti‐HCV E2 polyclonal antibody was a product of Biodesign International (Kennebunk, ME, USA). Anti‐HCV core mAb was purchased from Affinity BioReagents (Golden, CO, USA). Anti‐human CD81 mAb (clone JS81) and antihuman LDLR mAb (clone 15C8) were obtained from BD PharMingen and Oncogene, respectively. Rhodamine‐conjugated rabbit antigoat immunoglobulin G (IgG) and rabbit antigoat IgG coupled with fluorescein isothiocyanate (FITC) were from Jackson ImmunoResearch. FITC‐labelled goat antimouse IgG and bovine serum albumin (BSA) were supplied by Sigma (St. Louis, MO, USA). MEK1/2 inhibitor U0126 and rabbit polyclonal antibodies against c‐Raf, MEK1/2, phospho‐MEK1/2 (Ser217/221), MAPK/ERK, phospho‐MAPK/ERK (Thr202/Tyr204), p38 MAPK and phospho‐p38 MAPK (Thr180/Tyr182) were purchased from Cell Signalling Technology Inc. (Beverly, MA, USA). Rabbit polyclonal antibody specific for phospho‐Raf‐1 (Ser338) was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Dmethyl thiozolyl‐2′, 5′‐diphenylo‐2‐H‐tetrazolium bromide (MTT) assay kit was a product of Chemicon International (Temecula, CA, USA). L‐02 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco‐BRL, Rockville, MD USA) supplemented with 10% foetal bovine serum (FBS, HyClone, Logan, UT, USA), penicillin (100 units/mL) and streptomycin (100 µg/mL, Sigma) at 37 °C with 5% CO2.

Measurement of MAP kinases

To reduce constitutive kinase activity, L‐02 cells were maintained for 12 h in serum‐free DMEM for the following assays. HCV E2 protein (1 µg/mL) was mixed with 2 µg/mL HCV E2 mAb or HCV core mAb for 1 h at 37 °C. Cells were treated for 15 min with 1 µg/mL E2 protein, a mixture of E2‐E2 mAb or E2‐core mAb. For competition assays, cells were pretreated for 1 h with 4 µg/mL CD81 mAb, 4 µg/mL LDLR mAb, a combination of CD81 mAb and LDLR mAb or various concentrations of U0126 dissolved in dimethyl sulfoxide, washed twice with phosphate‐buffered saline (PBS), and exposed to 1 µg/mL E2 protein for an additional 15 min. In addition, cells were cultured for 48 h in medium containing 1% FBS plus different concentrations of E2 protein. After the indicated treatments, cells were harvested and were lysed for Western blot analysis to estimate the levels of MAP kinase activity.

Cell lysates were cleared by centrifugation at 13 400 g for 10 min. Equal amounts of protein extracts were subjected to 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes, followed by incubation overnight at 4 °C with rabbit polyclonal antibodies against c‐Raf, phospho‐Raf‐1, MEK1/2, phospho‐MEK1/2, MAPK/ERK, phospho‐MAPK/ERK, p38 MAPK and phospho‐p38 MAPK. Bands were developed with alkaline phosphatase‐conjugated goat antirabbit secondary antibody, nitroblue tetrazolium and 5‐bromo‐4‐chloro‐3‐indolyl‐phosphate substrates.

HCV E2‐cell interaction assay

Binding of the HCV E2 protein to L‐02 cells was detected by fluorescence‐activated cell sorting (FACS) assay, using a flow cytometer (EPICS®.XL, Coulter, Hialeah, FL, USA) with System software version 3.0 for acquisition and analysis. In the region of 1 × 106 cells, suspended in PBS containing 1% BSA, were incubated with 6 µg/mL E2 protein for 1 h at 37 °C. Cells were washed twice with PBS by centrifugation at 400 g for 5 min to remove unbound E2, were re‐suspended in 1% BSA‐PBS and were incubated with 8 µg/mL HCV E2 mAb for another 1 h. Cells were washed twice with PBS and were incubated with FITC‐labelled goat antimouse IgG for 1 h at 4 °C in the dark. After three washes with PBS, cells were fixed with 1% paraformaldehyde in PBS and were subjected to FACS analysis for E2 binding.

CD81 and LDLR at the surface of L‐02 cells were identified as previously described (Zhao et al. 2005). For binding inhibition assays, cells were incubated with 8 µg/mL CD81 mAb or LDLR mAb for 1 h at 37 °C, washed twice with PBS, and then were incubated with 6 µg/mL E2 protein for another 1 h. After two washes with PBS, cells were incubated with 8 µg/mL goat anti‐HCV E2 antibody for 1 h, washed twice with PBS, and then incubated with FITC‐conjugated rabbit antigoat IgG for 1 h at 4 °C in the dark. Binding of E2 protein to cells was analysed by flow cytometry.

Confocal microscopy

L‐02 cells were treated for 4 h with 6 µg/mL HCV E2 protein at 37 °C, were washed twice with PBS, and then were incubated with 8 µg/mL goat anti‐HCV E2 antibody and 4 µg/mL CD81 mAb overnight at 4 °C. For the localization of LDLR assay, cells were cultured for 48 h in DMEM without FBS and then were treated with E2 for 4 h at 37 °C, followed by incubation overnight at 4 °C with goat anti‐HCV E2 antibody and 4 µg/mL LDLR mAb. Cells were then washed three times with PBS and incubated with rhodamine‐conjugated rabbit antigoat IgG and FITC‐labelled goat antimouse IgG for 1 h at 4 °C in the dark. After being thoroughly washed with PBS, cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Cells were centrifuged, re‐suspended in PBS, and were seeded on poly L‐lysine‐coated glass coverslips. The coverslips were rinsed three times in PBS and were mounted on slides. Fluorescence images were photographed using a Leica TCS SP2 confocal microscope.

Detection of cell proliferation

L‐02 cells were trypsinized, suspended in 1% FBS‐DMEM at a density of 2 × 105 cells per millilitre, and were aliquoted into 96‐well tissue culture plates (0.1 mL per well). Cells were cultured in the medium containing varying concentrations of HCV E2 protein, BSA control or a mixture of HCV E2‐E2 mAb. After 48 h of culture, an MTT assay was carried out for detection of cell proliferation, according to the manufacturer's instructions. In brief, 5 mg/L MTT was added directly to the cells and specimens were incubated for another 4 h, followed by incubation in isopropanol with 0.04 N HCl for 30 min. Optical density (OD) was determined using an enzyme‐linked immunosorbent assay reader (Bio‐Rad). Data were presented as the mean OD and standard deviation from triplicate cultures. Statistical analysis was performed using Student's t‐test. Differences were considered to be statistically significant at P‐values < 0.05.

RESULTS

Modulation of both upstream and downstream MAP kinases by HCV E2

Enhancement of MAP kinase activity involves phosphorylation at relevant residues of each kinase. Raf kinases are an entry point to the MAPK/ERK pathway. In the case of Raf‐1 (also termed c‐Raf), the best‐studied isoform of Raf kinases, phosphorylation of serine 338, is a good indicator of Raf‐1 activation (Dhillon & Kolch 2002). Activation of MEK1/2 occurs through phosphorylation of two serine residues at positions 217 and 221. MAPK/ERK is activated by dual phosphorylation at threonine 202 and tyrosine 204. Raf phosphorylates and activates MEK1/2 and that, in turn, phosphorylates and activates MAPK/ERK. Here, Raf‐1‐MEK1/2‐MAPK/ERK signalling cascades initiated by HCV E2 protein were investigated.

Hepatitis C virus genotype 1a and 3 core proteins activated MEK1 and ERK1/2 MAP kinases, which contribute to neoplastic transformation of HCV‐infected liver cells (Giambartolomei et al. 2001). In human T cells, HCV core protein inhibits phosphorylation of ERK and MEK, and impairment of ERK/MEK kinase cascades results in inhibition of interleukin‐2 and interleukin‐2 receptor alpha gene transcription (Yao et al. 2001). Based on modulation of MAPK/ERK and p38 MAPK by HCV E2 protein, we wondered whether the upstream kinases Raf‐1 and MEK1/2 would be affected, which may be important to elucidate for MAPK pathways initiated by E2. Cellular responses towards the same signal may be different in different cell lines, so effects of E2 on Raf‐1, MEK1/2, MAPK/ERK and p38 MAPK were further investigated in these L‐02 cells. First, alterations of Raf‐1 and MEK1/2 were evaluated. Western blot analysis showed that levels of Raf‐1 and MEK1/2 phosphorylation were low in untreated cells maintained in serum‐free medium, and levels were increased in response to E2 treatment (Fig. 1a). Antibodies to E2 or to core protein were applied to ascertain whether increased phosphorylation would result from E2 treatment. Phosphorylation of Raf‐1 and MEK1/2 was impaired in cells treated with the E2‐E2 mAb, yet was unimpaired following E2‐core mAb treatment; this indicates activation of Raf‐1 and MEK1/2 induced by the E2.

Figure 1.

Figure 1

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Effects of HCV E2 protein on Raf, MEK1/2, MAPK/ERK and p38 MAPK. Serum‐starved L‐02 cells were treated for 15 min with E2 protein, E2‐E2 mAb mixture or E2‐core mAb mixture. Cells were lysed for detection of total and phosphorylated MAP kinases by Western blotting, using specific antibodies against Raf, phospho‐Raf, MEK1/2, phospho‐MEK1/2 (a), MAPK/ERK, phospho‐MAPK/ERK, p38 MAPK and phospho‐p38 MAPK (b).

Regulation of MAPK/ERK and p38 MAPK by E2 protein was then estimated. Figure 1b illustrates that phosphorylation of MAPK/ERK and p38 MAPK was weak in untreated cells, and that such phosphorylation was enhanced after exposure to E2 protein. Specificity of MAPK/ERK and p38 MAPK activation was also tested. Phosphorylation levels of MAPK/ERK and p38 MAPK were reduced in cells treated with the E2‐E2 mAb, and E2‐core mAb treatment resulted in almost undetectable changes in kinase phosphorylation, as compared to E2 stimulation, suggesting that E2 mAb specifically inhibits E2‐induced MAPK/ERK and p38 MAPK activation. Amounts of total Raf‐1, MEK1/2, MAPK/ERK and p38 MAPK were monitored as controls for protein loading. These results established that the HCV E2 protein up‐regulated upstream kinases (Raf‐1 and MEK1/2) and downstream kinases (MAPK/ERK and p38 MAPK) of MAPK pathways.

Inhibition of MAPK/ERK activation by U0126

Due to the complex nature of MAPK pathways and the multiplicity of kinases, use of inhibitors of MAP kinases is necessary for investigation of kinase cascades. U0126 is a highly specific and selective inhibitor of MEK1/2 and has been proposed for use as a powerful tool to study activation of MAPK/ERK by MEK1/2 (Favata et al. 1998). Influence of U0126 on regulation of the kinase cascade by the HCV E2 protein was thus estimated. For this purpose, L‐02 cells were pretreated for 1 h with U0126 at a final concentration of 1, 5 or 10 µm, and then were treated with E2. Our results showed that the MAPK/ERK phosphorylation induced by E2 protein was suppressed by U0126 and that such suppression was in a concentration‐dependent manner, as revealed that MAPK/ERK phosphorylation was notably suppressed in cells pretreated with 10 µm U0126 (Fig. 2). U0126 prevented HCV E2‐induced MAPK/ERK activation, which implied that such prevention might be due to interference of U0126 with the upstream kinase MEK1/2.

Figure 2.

Figure 2

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Inhibition of HCV E2‐induced MAPK/ERK phosphorylation by MEK1/2 inhibitor U0126. Serum‐starved L‐02 cells were pretreated for 1 h with the indicated concentrations of U0126 and were stimulated with E2 protein for an additional 15 min. Total and phosphorylated MAPK/ERK were analysed by Western blotting using the antibodies against MAPK/ERK and phospho‐MAPK/ERK.

Interaction of HCV E2 with CD81 and LDLR

Cell membrane receptors play a pivotal role in recognizing and transmitting extracellular stimuli to intracellular signal networks. We explored the roles of CD81 and LDLR in regulation of MAPK pathways by HCV E2 protein. By flow cytometry, these molecules were shown differently expressed at the surface of L‐02 cells (Fig. 3a). Mouse IgG served as isotype control to determine background fluorescence. E2 protein was tested for its ability to bind to L‐02 cells. Cells without E2 incubation (no antigen) were incubated with the E2 mAb and FITC‐labelled secondary antibody to establish background levels of binding. Figure 3b shows that a high percentage of cell‐bound E2 was detectable. Competition experiments were performed to determine whether such binding is in relation to CD81 and LDLR on the cells. Pretreatment with CD81 mAb or LDLR mAb reduced cell‐bound E2 (Fig. 3b), suggesting inhibition of E2 binding by CD81 mAb or LDLR mAb. Thus, E2 protein bound to L‐02 cells through its interaction with CD81 and LDLR.

Figure 3.

Figure 3

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Interaction of HCV E2 protein with cell receptors. Expression of CD81 and LDLR on L‐02 cells was analysed by flow cytometry. Mouse IgG served as an isotype control (a). Cells were incubated with or without E2 protein (no antigen), and E2 binding was assessed by FACS analysis. Cells were pretreated with CD81 mAb or LDLR mAb and were then incubated with E2 protein. Cell‐bound E2 was analysed (b). Cells were incubated with E2 protein, followed by incubation of CD81 mAb or LDLR mAb and goat anti‐E2 antibody. CD81 and LDLR expression and E2 distribution were monitored by confocal microscopy. Green fluorescence indicates localization of CD81 or LDLR and red fluorescence indicates distribution of E2. Merged images show colocalization of E2 protein with CD81, as well as with LDLR (c).

Cellular location of CD81 and LDLR and the distribution pattern of E2 protein were revealed by confocal microscopy. CD81 and LDLR were visualized with FITC‐labelled goat antimouse IgG, while E2 was visualized with rhodamine‐conjugated rabbit antigoat IgG. CD81 and LDLR (green fluorescence), were found to localize at the surface of cells (Fig. 3c). E2 (red fluorescence) was distributed both on the cell membrane plus in cytoplasmic regions. Areas of overlap indicate colocalization of E2 with CD81, as well as with LDLR. Low degree of overlap on cell membranes may be explained according to cytoplasmic distribution of E2. We speculated that internalization of HCV E2 protein occurred after incubation at 37 °C for 4 h. Under the same conditions, internalized HCV‐like particles have already been reported (Ludwig et al. 2004).

CD81‐ and LDLR‐mediated activation of MAP kinases

Hepatitis C virus E2 protein interacted with CD81 and LDLR on L‐02 cells. Next, levels of activity of the MAP kinases in response to the E2 treatment were analysed in cells with or without pretreatment with CD81 mAb or LDLR mAb. Figure 4a shows that the levels of Raf‐1 and MEK1/2 phosphorylation induced by E2 were reduced in cells pretreated with CD81 mAb or LDLR mAb. Combined pretreatment of CD81 mAb and LDLR mAb led to inhibition of Raf‐1 and MEK1/2 phosphorylation in a similar manner. Moreover, E2‐induced MAPK/ERK and p38 MAPK phosphorylation was decreased during blockage of CD81, as well as blockage of LDLR. As shown in Fig. 4b, levels of MAPK/ERK and p38 MAPK phosphorylation induced by E2 were reduced in cells pretreated with CD81 mAb or LDLR mAb. Cells were treated with CD81 mAb and LDLR mAb for comparison, to rule out potential negative effects of the mAbs. These results indicate that activation of Raf‐1, MEK1/2, MAPK/ERK and p38 MAPK induced by E2 was inhibited by CD81 mAb and LDLR mAb, implying that the mAbs might inhibit the activation of MAP kinases by HCV E2 protein competing with CD81 and LDLR.

Figure 4.

Figure 4

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Roles of CD81 and LDLR in regulation of MAP kinases by HCV E2 protein. Serum‐starved L‐02 cells were pretreated for 1 h with CD81 mAb, LDLR mAb or a combination of CD81 mAb and LDLR mAb, and received E2 stimulation for another 15 min. Levels of Raf, MEK1/2 (a), MAPK/ERK and p38 MAPK (b) were determined by Western blot analysis using the specific antibodies.

Dynamic phosphorylation of MAPK/ERK and p38 MAPK

MAPK/ERK phosphorylation in HepG2 cells is related to concentration and duration of HCV E2 stimulation (Zhao et al. 2001) and proliferation of HepG2 cells is driven by HCV core protein through activation of MAPK/ERK and p38 MAPK pathways (Erhardt et al. 2002). Considering the role of MAPK pathways in controlling cell population growth, we discussed whether dynamic phosphorylation of MAPK/ERK and p38 MAPK would be linked to L‐02 cell population growth. After incubation for 48 h with the HCV E2 protein, MAP kinase phosphorylation and cell proliferation were synchronously evaluated. Lysates from cells incubated with E2 at concentrations of 0, 1, 5, 10, 15 or 20 µg/mL were collected for detection of MAPK/ERK and p38 MAPK phosphorylation. Western blot analysis showed that 15 µg/mL E2 incubation resulted in strong phosphorylation of MAPK/ERK (Fig. 5a). Conversely, levels of p38 MAPK phosphorylation were high for 20 µg/mL E2 incubation, compared to the other concentrations.

Figure 5.

Figure 5

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Phosphorylation of MAPK/ERK and p38 MAPK and cell proliferation in response to HCV E2 treatment. L‐02 cells were cultured in 1% FBS‐DMEM in the presence of increasing concentrations of E2 protein. Up to 48 h, levels of MAPK/ERK and p38 MAPK in cell lysates were detected by Western blot analysis (a). Cells were cultured in medium containing the indicated concentrations of E2 protein, BSA or E2‐E2 mAb mixture. After 48 h, the MTT assay was performed to determine cell proliferation (b,c). Control indicates cells without treatment. Data are expressed as the mean and standard deviation of OD from triplicate cultures. Results are representative of three independent experiments.

Increased cell proliferation

Cell population growth was examined to characterize effects of HCV E2 protein on the biological behaviour of target cells. An MTT assay was carried out to detect proliferation of L‐02 cells incubated for 48 h with E2 or control antigen BSA at final concentrations of 0, 1, 5, 10, 15 or 20 µg/mL. Cell proliferation was found to be enhanced in the presence of E2 and such enhancement was not driven in a concentration‐dependent manner (Fig. 5b). The peak level of cell proliferation was observable for incubation with E2 at a concentration of 15 µg/mL. In contrast to E2, BSA incubation gave rise to a slight increase in cell proliferation. Statistical analysis demonstrated that levels of cell proliferation with 10, 15 or 20 µg/mL E2 were significantly higher than those without E2 incubation (P < 0.05). To determine whether increase in cell proliferation was driven by E2, the effect of the E2 mAb for such an increase was estimated. E2 protein was mixed with the E2 mAb for 1 h and then the cell preparation was incubated for 48 h. As shown in Fig. 5c, levels of cell proliferation were markedly reduced during E2‐E2 mAb incubation as compared to 15 µg/mL E2 (P < 0.05), suggesting that increased cell proliferation induced by E2 was abolished by 10 µg/mL E2 mAb. Accordingly, we believe that enhancement of L‐02 cell proliferation is driven by HCV E2 protein rather than other component(s).

DISCUSSION

Molecular mechanisms underlying HCV‐related liver diseases have not been elucidated thoroughly. As with numerous other viruses, HCV may exert its pathogenic effects through interference with signalling pathways. Modulation of signalling cascades by HCV core protein and nonstructural proteins is proven to account for development of disorders associated with HCV infection. Current data need to be reinforced regarding early signalling events triggered by HCV envelope proteins. Following on our previous work, we have addressed further the contribution of MAPK signalling pathways triggered by the HCV E2 protein to the proliferation of human liver cells. Much improvement is made in the present study, including extended investigations on upstream kinases Raf‐1 and MEK1/2, continuity of the kinase cascades and association between dynamic phosphorylation of MAPK/ERK and p38 MAPK and cell population growth. The colocalization of E2 with CD81 as well as LDLR is noted here.

HCV E2 protein is involved in mediating HCV binding to host cells and eliciting host immune responses. HCV E2 protein expressed in Escherichia coli or in eukaryotic cells, HCV‐like particles and chimaeric HCV envelope proteins have been successively developed to explore the features of E2 activity (Flint et al. 2000; Triyatni et al. 2002; Cocquerel et al. 2003). Recently, pseudoparticles consisting of unmodified HCV envelope glycoproteins assembled on retroviral core particles, and a cell culture system that allows efficient amplification of HCV, have been proposed as tools for the study of HCV interaction with candidate cellular receptors (Cocquerel et al. 2006). We focused on the modulation of MAPK pathways by the highly purified HCV E2 protein expressed in mammalian cells. Although interaction of E2 protein with target cells could not completely mimic the natural process of HCV infection, E2 can be used as an available biological tool to reveal early signalling events following the interaction. MAPK signalling cascades have been disrupted by HCV core protein with synergistic stimuli (Hayashi et al. 2000; Giambartolomei et al. 2001). Our results have established that E2 itself triggers MAPK signalling pathways and promotes liver cell population growth.

Inhibition of interferon‐inducible double‐stranded RNA‐activated protein kinase by HCV E2 protein has been suggested to be responsible for interferon resistance (Taylor et al. 1999). In HepG2 cells, HCV E2 protein and human immunodeficiency virus gp120 protein acted collaboratively to activate p38 MAPK and tyrosine phosphatase SHP2 (Balasubramanian et al. 2003) and activation of MAPK/ERK has been induced by HCV E2 protein in human hepatic stellate cells (Mazzocca et al. 2005). Human liver is a direct target organ for HCV infection. Here, upstream and downstream kinases of MAPK pathways were investigated in human liver cell line L‐02. Our data show that HCV E2 protein is an effective stimulator for MAPK pathways. Levels of Raf‐1, MEK1/2, MAPK/ERK and p38 MAPK phosphorylation are high upon E2 treatment compared to the basal levels of kinase phosphorylation. The specific E2 mAb reduced the phosphorylation levels of MAP kinases, suggesting that increased phosphorylation of Raf‐1, MEK1/2, MAPK/ERK and p38 MAPK is indeed induced by E2. Although presence of antibodies to E2 protein is not sufficient for viral clearance or prevention of HCV challenge, such antibodies might play an active role in down‐regulation of MAPK pathways. Furthermore, prevention of E2‐induced MAPK/ERK activation by MEK1/2 inhibitor U0126 has indicated that the kinase cascades were modulated by E2; in this way, kinase cascades of MAPK signalling pathways were continuously affected by presence of HCV E2 protein.

Interaction of HCV E2 protein with CD81 on human B cells has enhanced expression of activation‐induced cytidine deaminase and has stimulated production of tumour necrosis factor α (Machida et al. 2005). Engagement of CD81 by a combination of HCV E2 protein and an anti‐CD81 mAb triggered the JNK pathway and led to preferential proliferation in naive (CD27‐) B cell subset (Rosa et al. 2005). Interplay between HCV E2 protein and L‐02 cells was defined and such interplay provided the E2 signal for MAPK pathways. Our data have shown that E2 binding was mediated by CD81 and LDLR on cell surfaces. In response to E2 treatment, phosphorylation levels of Raf, MEK1/2, MAPK/ERK and p38 MAPK increased. Consistent with interactions of E2 with CD81 as well as LDLR, levels of Raf, MEK1/2, MAPK/ERK and p38 MAPK phosphorylation induced by E2 were decreased by blockage of CD81, as well as blockage of LDLR. Hence, up‐regulation of MAPK signalling pathways appeared to be partially mediated by CD81 and LDLR. However, whether CD81 and LDLR have tyrosine kinase activity or whether any potential adaptor proteins are linked to MAPK pathways remains to be studied. Efforts would be worthwhile to investigate downstream effectors of CD81 and LDLR. Human scavenger receptor class B type I and liver/lymph node‐specific intercellular adhesion molecule‐3‐grabbing integrin are candidate receptors for HCV (Scarselli et al. 2002; Gardner et al. 2003). Roles of these receptors in MAPK signalling pathways initiated by HCV E2 protein have not been studied in our experiments.

Interestingly, L‐02 cell proliferation was not promoted by HCV E2 protein in a concentration‐dependent manner. We found that the 15 µg/mL E2 incubation resulted in the peak level of cell proliferation. The high MAPK/ERK/p38 (SAPK) ratio has been reported to facilitate cancer cell population growth, whereas high p38 (SAPK)/MAPK/ERK ratio predicted cell population growth arrest (Aguirre‐Ghiso et al. 2003). Association between L‐02 cell proliferation and dynamic changes in MAPK/ERK and p38 MAPK phosphorylation was then discussed. Our results showed that strong phosphorylation of MAPK/ERK was induced in response to the 15 µg/mL E2 incubation, and that the highest level of p38 MAPK phosphorylation was observed following the 20 µg/mL E2 incubation, which might be relevant to explanation of difference in L‐02 cell proliferation with the different concentrations of E2 protein. Abnormal regulation of MAPK pathways is proven to be a molecular basis of human tumourigenesis such as in skin tumours, gastric carcinomas, breast cancers, and head and neck squamous cell carcinomas (Hildesheim et al. 2002; Kanai et al. 2003; Lui et al. 2003; Ma et al. 2003). Over‐proliferation favours acquisition of growth advantage for HCV‐infected cells, resulting in viral persistence leading to chronic infection that underlies establishment and progression of HCV‐related diseases. Inhibition of signalling pathways is implicated for the control of viral infection. Chemical inhibitors of host signalling pathways facilitate control of poxvirus infection (Yang et al. 2005) and STAT1‐dependent and ‐independent pathways play critical roles in the control of primary dengue virus infection (Shresta et al. 2005). Persistent NF‐κB activation in herpes simplex virus‐1‐infected cells plays a positive role in promoting efficient viral replication (Amici et al. 2006) and the anti‐HCV effect of interferon‐γ has been linked to control of HCV replication through the Ras‐MAPK pathway (Huang et al. 2006). These results provide a potential strategy for control of viral infection by affecting signalling molecules. Our findings raise the possibility that target cells may be influenced as a result of MAPK pathways triggered by the E2, which likely represents an important early signalling event following HCV infection. Therefore, regulation of MAPK signalling by E2 protein has implications for prevention and control of HCV infection.

ACKNOWLEDGEMENTS

We are very grateful to Drs Michael Houghton, Christine Dong and Shirley Wong (Chiron Corporation) for providing the HCV E2 protein and the E2 mAb. We also thank Lecturer Ying Tang (Department of Mathematics and Physics, Second Military Medical University, Shanghai, China) for technical assistance with confocal microscopy. This work was supported by the Natural Science Foundation of China (30500021) (to L.J.Z.) and the Natural Science Foundation of China (30670089) (to Z.T.Q.).

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