Hepatitis C virus (HCV) is the major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, and can be involved in very long chronic infections up to 30 years or more. Therefore, it has been speculated that HCV possesses mechanisms capable of modulating host defense systems such as innate and adaptive immunity. To investigate this virus-host interaction, we generated HCV replicons containing various HCV structural proteins and then analyzed the sensitivity of replicon-containing cells to the apoptosis-inducing agent, TRAIL. TRAIL-induced apoptosis was monitored by cleavage of procaspase-3 and procaspase-9 as well as that of their substrate poly(ADP-ribose) polymerase. TRAIL-induced apoptosis was inhibited in cells expressing HCV E2. Moreover, expression of HCV E2 enhanced the colony forming efficiency of replicon-containing cells by 25-fold. Blockage of apoptosis by E2 seems to be related to inhibition of TRAIL-induced cytochrome c release from the mitochondria. Based on these results, we propose that E2 augments persistent HCV infection by blocking host-induced apoptosis of infected cells.

Cellular antiviral responses are capable of blocking effective viral infection, but many viruses possess mechanisms that counteract these antiviral activities (1). Within the host, broad-spectrum innate immunity and infection-specific adaptive immunity play major roles in virus clearance at the early and late phases, respectively (2). Both innate and adaptive immune responses rely on apoptosis of virus-infected cells (3), which is triggered intracellularly by infection-induced modification of cellular activities (4) or by cytokines produced by the virus-infected cells (autocrine) or by other cells in the vicinity (paracrine) (3, 5).

Two different apoptotic pathways have been extensively studied. In the first pathway, cytotoxic agents or intracellular signals induce mitochondrial changes leading to the release of cytochrome c from the mitochondrial membrane. This effect is controlled by members of the Bcl-2 protein family, which includes both antiapoptotic (Bcl-2 and Bcl-xL) and proapoptotic (bad, bax, and bak) molecules (6, 7, 8, 9). The second pathway is mediated by a signal transduction cascade driven by death ligands, which are members of the TNF family including TNF, Fas/CD95/Apo1 ligand, TRAIL/Apo2 ligand, and TWEAK/DR3/Apo3 ligand (10).

Apoptotic progression depends on the cell type and the surrounding conditions. Death ligand-driven apoptosis begins when the ligand interacts with the corresponding “death receptor” on the cell surface. Specifically, the TRAIL/Apo2 ligand (referred to in this study as TRAIL), which preferentially kills transformed cells (11, 12), induces apoptosis by interacting with its death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). Following receptor binding, caspase-8 is activated, whereupon signal transduction by the receptors diverges into two pathways for several steps and merges again at the caspase-3/7 activation stage. In terms of TRAIL signaling, cells can be divided into two subgroups: type I, which have high levels of caspase-8, and type II, which have low levels of caspase-8. In type I cells, such as a number of lymphoid cell lines, activated caspase-8 directly cleaves and activates procaspase-3/7 (13). In type II cells, such as hepatocyte cell lines, caspase-8 activation is not sufficient to directly activate caspase-3/7; these cells use a mitochondrial signal amplification loop to transduce the apoptotic signal from caspase-8 to caspase-3/7. In these cells, caspase-8 cleaves the cytoplasmic Bid protein to produce a Bid fragment that triggers mitochondrial signal transduction (14). TRAIL-induced apoptosis of Huh-7, a human hepatoma cell line, is known to occur through the type II pathway; overexpression of Bcl-xL, which blocks apoptosis through the mitochondria, inhibits TRAIL-induced apoptosis (15). In this study, we used the TRAIL-induced apoptosis of Huh-7 cells as a model system for investigating antiviral signaling.

Viruses often ensure their own survival by blocking host anti-infective apoptotic mechanisms (16, 17, 18, 19) with a variety of viral proteins that modulate different parts of the death signaling pathways (6). For instance, the adenoviral E3-complex (composed of 10.4, 14.5, and 6.7 kDa proteins) down-regulates death receptors such as Fas, DR4, and DR5 to reduce the antiviral effects of Fas ligand and TRAIL signaling (20, 21, 22). Similarly, expression of the herpesvirus E8 protein protects cells from Fas- and TNFR1-induced apoptosis through an interaction with the death effector domain-containing protein (17).

Hepatitis C virus (HCV)3 is the major causative agent for hepatitis and hepatocellular carcinoma (23, 24). One of the characteristics of HCV is its strong propensity for persistent infection, which results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (25). Epidemiological studies have suggested that ∼80% of acute HCV cases develop into chronic infection (26, 27). This high incidence of chronic HCV infection suggests that the virus generates proteins that actively block the antiviral functions of the host.

HCV is a positive sense RNA virus encoding 3 virion structural proteins (core, E1, and E2) and 7 nonstructural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) responsible for viral RNA replication and/or modulation of host functions (28). Several HCV proteins have been suggested to participate in modulating host cellular activities. For instance, the core protein is thought to induce or block apoptosis, transform cells, and modulate transcription and translation by interacting with cellular proteins including 14-3-3, apolipoprotein AII, TNFR1, and the lymphotoxin receptor (29, 30, 31, 32). Very recently, the core protein was shown to enhance TRAIL-mediated apoptosis of human hepatoma cell line Huh-7 (33). Several other HCV proteins such as nonstructural NS3, NS5A, and NS5B were also suggested to induce apoptosis of cells (34, 35). Structural protein E2 and nonstructural protein NS5A are thought to block RNA-dependent protein kinase R (PKR), which is responsible for IFN-mediated apoptosis through protein-protein interactions (36, 37), and nonstructural proteins NS3 and NS4B stimulate transformation of NIH3T3 cells (38, 39). These previous investigations were performed in heterologous cell lines with ectopic expression of the viral proteins. A new approach has become possible by the development of a HCV replicon system using an artificial subgenomic HCV RNA (40). The HCV replicon RNAs replicate autonomously to high levels upon transfection into human hepatoma cell line Huh-7, thus mimicking viral proliferation in a cell (41). This relatively new model should prove useful for examination of the roles of viral proteins during infection and blocking of antiviral host activities.

To investigate the roles of viral proteins during infection, we generated artificial replicons expressing different HCV proteins (core, E1, E2, p7, and NS2) along with the minimal regions required for viral RNA replication (the protein-coding region from nonstructural proteins NS3 to NS5B and the 5′ and 3′ nontranslated regions). We then investigated the effects of proapoptotic TRAIL on various replicon-containing cells. Among the tested HCV proteins, E2 blocked apoptosis of replicon-containing cells by interrupting the mitochondrial-based death signaling pathway. The blockage of apoptosis by E2 seems to be related to a reduced cytochrome c release from the mitochondria. Together, these results suggest that E2 plays a key role in persistent HCV infection and/or hepatocellular carcinoma development.

Plasmid FK included a full-length replicon encoding all viral proteins. For generation of this plasmid, the Con1 plasmid (42) was PCR amplified with oligonucleotides H95 (5′-CTCATGAAGTTGGCCGCACTG-3′) and H96 (5′-TGCTCTAGAACTAGAGGAGTCGCCACCCCTGCC-3′; adds an XbaI site). The resultant-containing PCR fragment was digested with BsiWI and XbaI and inserted into the XbaI and NruI sites of NK/H(1-389)/P (43) along with the NruI/BsiWI fragment of the Con1 plasmid (42). FK-C399T+2 (UAA), which expresses only the HCV nonstructural proteins due to introduction of a termination codon, was generated by the addition of two nucleotides at HCV nt 399 in plasmid FK. FK-core (UAA) was constructed by ligation of the FK ClaI/NotI fragment along with the ClaI/XbaI and NotI/XbaI fragments of a PCR product. The product was generated by amplifying template DNA FK with oligonucleotides H166 (5′-GCGTAGGTCGCGCAATTTGGG-3′) and H164 (5′-TGCTCTAGACT TAAGCGGAAGCTGGGATGGTC-3′) containing ClaI and XbaI sites or H165 (5′-TGCTCTAGATATGAAGTGCGCAACGTATC-3′) and H167 (5′-CAG CCAAACCAGTTGCCTTG-3′) containing NotI and XbaI sites. FK-E1 (UAA) was constructed in the same manner using a PCR fragment amplified from FK using primers H166 and H168 (5′-TGCTCTAGACTTACCCGTCAACGCCGGCAAAG-3′) containing ClaI and XbaI sites, or H169 (5′-TGCTCTAGAGGAACCTATGTGACAGGGG-3′) and H167 (5′-CAGCCAAACCAGTTGCCTTG-3′) containing NotI and XbaI sites. FK-E2 (UAA) was constructed by ligation of the FK BsiWI/NotI fragment to the NotI/XbaI and BsiWI/XbaI fragments of the PCR product. PCR product was generated by amplification of FK with primers H170 (5′-CGTGCTGCTTCTTAACAACAC-3′) and H171 (5′-TGCTCTAGACTTAGGCCTCAGCTTGAGCTATC-3′) containing NotI and XbaI sites or H172 (5′-TGCTCTAGAGCCCTAGAGAACCTGGTGG-3′) and H173 (5′-CAGTCCCGCAGTGGGGTGAG-3′) containing BsiWI and XbaI sites. FK-p7 (UAA) was constructed as described, using the PCR fragment generated with primers H170 and H174 (5′-TGCTCTAGACTTAGGCGTATGCTCGTGGTGGTAAC-3′) containing NotI and XbaI sites or H175 (5′-TGCTCTAGAATGGACCGGGAGATGGCAG-3′) and H173 containing BsiWI and XbaI sites.

To generate run-off transcripts of HCV replicons, plasmids were linearized with ScaI. The linearized DNAs were then phenol-chloroform extracted and precipitated with ethanol. Transcription reactions were performed with T7 RNA polymerase (Stratagene) as described by the manufacturer. After 2 h incubation at 37°C, 40 U of additional T7 RNA polymerase were added, and the reaction mixture was incubated for another 2 h, after which the RNA transcripts were prepared as described by Kim et al. (43).

Huh-7 human hepatoma cells were routinely grown in monolayer cultures in DMEM (Invitrogen Life Technologies) with 10% FBS (HyClone Laboratories), penicillin and streptomycin. Cells were electroporated as previously described (43).

For induction of apoptosis, cells were treated with 200 ng/ml TRAIL for 1–2 h. Cells were collected and washed twice with cold PBS and lysed by sonication for Western blot analysis. The protein content of all extracts was measured with the Coomassie Plus Protein Assay Reagent kit (Pierce).

Morphological changes in the nuclear chromatin of cells before and after treatment of TRAIL (200 ng/ml) for 3 h were detected by staining with 2 μg/ml Hoechst 33342 fluorochrome (Molecular Probes), followed by examination under a fluorescence microscope (Zeiss).

Proteins were resolved by 10 or 13.5% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham). The membrane was blocked overnight with 5% skim milk in TBS buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Tween 20) and then incubated with mAbs against actin (1/500), poly(ADP-ribose) polymerase (PARP; 1/200), Bcl-xL (1/1000), Bcl-2 (1/500), cytochrome c (1/500), or a polyclonal Ab against caspase-9 (1/1000) for 5 h. A monoclonal anti-human actin Ab was used as the control. HRP-conjugated anti-mouse or anti-rabbit IgGs were used as secondary Abs (1/5000), and bands were visualized by ECL according to the supplier’s instructions (Amersham).

Cellular caspase-3 activities were assayed with the modified Fluorometric CaspASE Assay System (Promega). One hundred micrograms of protein extracted from TRAIL-treated and untreated replicon-containing cells were assayed for caspase-3 activity by measuring their ability to cleave the fluorogenic substrate Ac-DEVD (acetyl-Asp-Glu-Val-Asp)-7-amino-4-methylcoumarin (AMC). Free AMC accumulation resulting from cleavage of the aspartate-AMC bond was monitored for 30 min using a spectrofluorometer (Cary Eclipse; Varian) at 360 nm excitation and 460 nm emission wavelengths. The assay specificity was evaluated by repeating the experiment in the presence of the caspase-3 inhibitor Ac-DEVD-aldehyde. Each assay was performed three times.

FACS analysis with or without cells being permeabilized to the anti-DR4 Ab (mAb to DR4 conjugated with biotin; IMGENEX) and anti-DR5 Ab (ALEXIS Biochemicals) to monitor total DR4/DR5 and DR4/DR5 on the cell surface, respectively. Quantities of DR4 and DR5 were analyzed by using the CellQuest analysis program (BD Biosciences). Mean fluorescence intensity values reflect ratios of replicon-containing cells to control Huh-7 cells. No difference in intensity of fluorescence among various replicon-containing cells was detected when a mouse IgG mAb conjugated-PE was used as an isotype control (data not shown). Approximately 5 × 105 cells were used in each FACS analysis.

Cells were collected, washed, and resuspended in fractionation buffer containing 250 mM sucrose, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and a protease inhibitor mixture. Cells were incubated for 10 min at 4°C, Nonidet P-40 was added to a final concentration of 10%, the mixtures were incubated for an additional 10 min at 4°C and then centrifuged at 1000 × g for 10 min at 4°C to remove intact cells and nuclei. Supernatants were centrifuged at 10,000 × g at 4°C for 25 min to separate mitochondrial and cytosolic fractions, and 30 μg of protein from each sample was subjected to 13.5% SDS-PAGE followed by Western blotting using an anti-cytochrome c mAb (BD Pharmingen).

To investigate the effect of HCV structural proteins on apoptosis of host cells, we generated Huh-7 cells containing and expressing modified replicons (Fig. 1). The tested replicons contained RNAs encompassing the entire HCV genome, but each expressed different proteins, including the structural proteins p7 and NS2, as well as the nonstructural proteins from NS3 to NS5B. These replicons were generated from a full-length HCV replicon (Fig. 1,A, shown as FK) by introducing artificial stop codons at the end of the genes encoding the core, E1, E2, p7, or NS2 proteins. Translation of the various proteins was directed by different internal ribosomal entry site (IRES). HCV structural proteins, neo gene product, and HCV nonstructural proteins from NS3 to NS5B were translated by the HCV IRES, the polioviral IRES, and the IRES of encephalomyocarditis virus, respectively (Fig. 1,A). Huh-7 cells were transfected with the various RNA replicons via electroporation, and cells supporting the continuous replication of HCV replicons were selected with G418. The selected Huh-7 colonies were pooled and cultivated for further analysis or fixed on the cell culture dish, stained with crystal violet, and counted (see Fig. 6).

FIGURE 1.

Generation of Huh-7 cells containing various replicons. A, Schematic diagram of HCV replicons generated from a full-length HCV replicon (FK). All of the constructs contain the same coding sequences for structural and nonstructural proteins, but express different structural proteins as designated by stop codons artificially introduced in the structural region of HCV. Solid lines and gray lines depict the translated and untranslated regions, respectively. B, Detection of HCV proteins in G418-resistant Huh-7 colonies. To monitor the expression levels of viral proteins in Huh-7 cells containing HCV replicons, Western blot analysis was performed using a mAb against E2 and polyclonal Abs against core and NS5B. A mAb against human actin was used for control purposes.

FIGURE 1.

Generation of Huh-7 cells containing various replicons. A, Schematic diagram of HCV replicons generated from a full-length HCV replicon (FK). All of the constructs contain the same coding sequences for structural and nonstructural proteins, but express different structural proteins as designated by stop codons artificially introduced in the structural region of HCV. Solid lines and gray lines depict the translated and untranslated regions, respectively. B, Detection of HCV proteins in G418-resistant Huh-7 colonies. To monitor the expression levels of viral proteins in Huh-7 cells containing HCV replicons, Western blot analysis was performed using a mAb against E2 and polyclonal Abs against core and NS5B. A mAb against human actin was used for control purposes.

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FIGURE 6.

The colony forming efficiencies of cells containing the various HCV replicons. Replicon-containing cells were selected with G418 for 15 days. Colonies were visualized by crystal violet staining and counted.

FIGURE 6.

The colony forming efficiencies of cells containing the various HCV replicons. Replicon-containing cells were selected with G418 for 15 days. Colonies were visualized by crystal violet staining and counted.

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To investigate the expression of the replicon-encoded viral proteins, Western blot analyses were performed using total cell extracts from the replicon-containing cells. The amount of total protein in the extracts was normalized against the corresponding actin levels. Successful expression of the core, E2, and NS5B viral proteins was detected by the corresponding Abs (Fig. 1,B). The level of core in FK-core (UAA)-containing cells was lower than that in other core-expressing cells, even though the level of NS5B was the same in all replicon-containing cells (Fig. 1 B; compare lane 3 with lanes 4–6). The reason for this result remains to be elucidated. One possible explanation is that coexpression of E1 and core may stabilize core through a protein-protein interaction.

To investigate the effects of the various HCV proteins on apoptosis, transfected and nontransfected Huh-7 were treated with 200 ng/ml TRAIL in the culture media. Morphological changes of cells (nuclear membrane ruffling, plasma membrane blebbing, and cell detachment) were detected in TRAIL-treated control Huh-7 cells from 1 h posttreatment. Similar morphological changes were detected in TRAIL-treated Huh-7 cells containing replicons FK-C399T+2, FK-core (UAA), and FK-E1 (UAA). In contrast, Huh-7 cells containing E2-expressing replicons FK-E2 (UAA), FK-p7 (UAA), and FK showed dramatically reduced morphological changes (data not shown). Moreover, TRAIL strongly induced nuclear condensation and DNA fragmentation, which are hallmarks of apoptosis, in control Huh-7 cells (Fig. 2,A, image 1b) and in Huh-7 cells containing replicons FK-C399T+2 (UAA), FK-core (UAA), and FK-E1 (UAA) that do not express E2 (Fig. 2,A, image 2b, 3b, and 4b). In contrast, markedly reduced levels of nuclear condensation and DNA fragmentation were observed in the Huh-7 cells containing E2-expressing replicons FK-E2 (UAA), FK-p7 (UAA), and FK at the same conditions (Fig. 2 A, image 5b, 6b, and 7b). This may indicate that E2 decreases TRAIL-induced apoptosis of Huh-7 cells.

FIGURE 2.

TRAIL-induced activation of apoptotic signaling in replicon-containing Huh-7 cells. A, Distribution patterns of DNAs in cells before (a) and after (b) treatment of TRAIL were observed by a fluorescence microscope. After treatment of Huh-7 cells containing various replicons with 200 ng/ml TRAIL for 3 h, chromatin condensation and fragmentation were monitored by Hoechst 33342 staining. B and C, Activation of caspase-9 was monitored by the cleavage of procaspase-9 (α-caspase-9). The level of total proteins was monitored by the amount of actin (α-Actin). The Huh-7 cell extracts depicted are before (B) and after (C) TRAIL treatment. In B, a negative control obtained from Huh-7 cells (lane 1) lacking both a replicon and TRAIL treatment is shown. The replicons are designated at the top of B and C. Cells were treated with 200 ng/ml TRAIL for 2 h, and procaspase-9 band intensities were assessed with Scion Image (NIH Image) and depicted as percentiles (%). D, Quantitative analysis of caspase-3 activity in cells containing the different replicons. Activities of caspase-3 was measured in cells containing the various replicons before (time 0) and after treatment of cells with 200 ng/ml TRAIL for 1 (time 1) and 2 h (time 2), as reflected by cleavage of the caspase-3 substrate DEVD-AMC in the presence (odd-numbered lanes) or absence (even-numbered lanes) of caspase inhibitor DEVD-aldehyde. E and F, Effect of long-term treatment of TRAIL and etoposide on PARP. Cells were treated with 50 ng/ml TRAIL (E) and/or 200 μM etoposide (F) for 18 and 36 h. Cleavage of PARP (α-PARP) was monitored as an indicator of caspase-3 activation.

FIGURE 2.

TRAIL-induced activation of apoptotic signaling in replicon-containing Huh-7 cells. A, Distribution patterns of DNAs in cells before (a) and after (b) treatment of TRAIL were observed by a fluorescence microscope. After treatment of Huh-7 cells containing various replicons with 200 ng/ml TRAIL for 3 h, chromatin condensation and fragmentation were monitored by Hoechst 33342 staining. B and C, Activation of caspase-9 was monitored by the cleavage of procaspase-9 (α-caspase-9). The level of total proteins was monitored by the amount of actin (α-Actin). The Huh-7 cell extracts depicted are before (B) and after (C) TRAIL treatment. In B, a negative control obtained from Huh-7 cells (lane 1) lacking both a replicon and TRAIL treatment is shown. The replicons are designated at the top of B and C. Cells were treated with 200 ng/ml TRAIL for 2 h, and procaspase-9 band intensities were assessed with Scion Image (NIH Image) and depicted as percentiles (%). D, Quantitative analysis of caspase-3 activity in cells containing the different replicons. Activities of caspase-3 was measured in cells containing the various replicons before (time 0) and after treatment of cells with 200 ng/ml TRAIL for 1 (time 1) and 2 h (time 2), as reflected by cleavage of the caspase-3 substrate DEVD-AMC in the presence (odd-numbered lanes) or absence (even-numbered lanes) of caspase inhibitor DEVD-aldehyde. E and F, Effect of long-term treatment of TRAIL and etoposide on PARP. Cells were treated with 50 ng/ml TRAIL (E) and/or 200 μM etoposide (F) for 18 and 36 h. Cleavage of PARP (α-PARP) was monitored as an indicator of caspase-3 activation.

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The TRAIL-induced apoptosis of replicon-containing cells was confirmed by analyzing proteolytic cleavage of PARP and caspase-9, which are indicators of caspase-3/7 activation and induction of mitochondrial-based apoptosis, respectively (Fig. 2). Before TRAIL treatment, the levels of PARP and procaspase-9 were the same in Huh-7 cells irrespective of the presence and type of replicon (Fig. 2, B and C). Following TRAIL treatment (200 ng/ml) for 2 h, the majority of PARP proteins were cleaved in control Huh-7 cells (Fig. 2,C, lane 2, top). Note that residual intact PARP could be detected. Huh-7 cells containing replicons FK-C399T+2 (UAA), FK-core (UAA), and FK-E1 (UAA), which do not express E2, showed stronger PARP cleavage following TRAIL treatment; no intact PARP was detected in these cells (Fig. 2,C, lanes 3–5, top). In contrast, PARP cleavage was greatly inhibited by E2, as seen in cells containing the E2-expressing replicons, FK-E2 (UAA), FK-p7 (UAA), and FK (Fig. 2,C, lanes 6–8, top). Similar patterns of caspase-9 activation were detected when cleavage of procaspase-9 was investigated (Fig. 2,C, middle). Following TRAIL treatment, about half of the procaspase-9 proteins remained intact in control cells (Fig. 2,C, lane 2) and those expressing the E2-null replicons FK-C399T+2 (UAA), FK-core (UAA), and FK-E1 (UAA) (Fig. 2,C, lanes 3–5, middle). In contrast, most of the procaspase-9 proteins remained intact in TRAIL-treated cells containing the E2-expressing replicons FK-E2 (UAA), FK-p7 (UAA), and FK (Fig. 2 C, lanes 6–8, middle). The relative amounts of intact caspase-9 in the cells containing various replicons post-TRAIL treatment are shown as percentile, considering the amount of caspase-9 in Huh-7 cells before TRAIL treatment as 100%.

The progress of apoptosis was also monitored by quantification of activated caspase-3. For quantitative analysis of caspase-3 activation, we measured caspase-3 protease activity in cell extracts of replicon-containing cells for 1 and 2 h following TRAIL treatment (Fig. 2,D). Cells containing replicon FK-C399T+2 showed an ∼2-fold increase in caspase-3 activity compared with control Huh-7 cells (Fig. 2,D, compare lane 3 with lane 9 and lane 5 with lane 11). Caspase-3 activity was further increased in cells expressing the core protein (FK-core (UAA) and FK-E1 (UAA)) (Fig. 2,D, compare lane 9 with lane 15 and lane 21). Interestingly, Huh-7 cells containing E2-expressing replicons FK-E2 (UAA), FK-p7 (UAA), and FK showed much lower caspase-3 activity than cells containing the E2-null replicons FK-C399T+2, FK-core (UAA), and FK-E1 (UAA) (Fig. 2,D; compare lanes 27, 33, and 39 with lanes 9, 15, and 21; compare lanes 29, 35, and 42 with lanes 11, 17, and 23). Moreover, the levels of activated caspase-3 in E2-containing cells were considerably lower than activated caspase-3 in control Huh-7 cells (Fig. 2 D; compare lanes 27, 33, and 39 with lane 3; compare lanes 29, 35, and 42 with lane 5). The data strongly suggest that the E2 protein of HCV inhibits TRAIL-induced apoptosis.

To investigate long term effects of TRAIL treatment on Huh-7 cells containing replicons, cleavages of PARP in Huh-7, Huh-7 cells with FK-E1 (UAA), and FK-E2 (UAA) were observed after treatment of TRAIL (50 ng/ml) for 18 or 36 h (Fig. 2,E). Reduced levels of intact PARP were observed in all cells after TRAIL treatment for 18 h (Fig. 2,E, lanes 2, 5, and 8). Most of intact PARP disappeared in the cells lacking E2 after treatment of TRAIL for 36 h (Fig. 2,E, lanes 3 and 6). In contrast, a large portion of PARP remained intact in the cells even after TRAIL treatment for 36 h (Fig. 2 E, lane 9). These results indicate that E2 protects cells from apoptosis induced by long term treatments of TRAIL.

Antiapoptotic effect of E2 was further analyzed by using another apoptosis-inducing agent etoposide, which is known to induce apoptosis through a mitochondria-mediated pathway (44). Similarly to TRAIL effect, treatment of etoposide (200 μM) for 18 h induced cleavage of PARP in Huh-7 cells regardless of the presence and type of replicons (Fig. 2,F, lanes 2, 5, and 8). Upon treatment of etoposide for 36 h, almost all of PARP proteins were cleaved in control cells (Fig. 2,F, lane 3) and cells expressing the E2-null replicon FK-E1 (UAA). Curiously, higher level intact PARP was detected in Huh-7 cells containing E2-expressing replicon FK-E2 (UAA) after treatment of etoposide for 36 h as compared with levels after treatment of etoposide for 18 h (Fig. 2 F, compare PARP on lane 9 with lane 8). The data indicate that E2 protects cells from apoptosis induced by treatments of etoposide.

The FK-E2 (UAA) replicon expresses not only E2, but also core and E1. To rule out the possibility of cooperative antiapoptotic activity of E2 with core and/or E1, we constructed replicon FK-OE2 (UAA) (Fig. 3,A) expressing full-length E2 fused with the C-terminal region of E1 (the signal sequence for E2). The cleavage pattern of PARP by TRAIL treatment of Huh-7 cells containing FK-OE2 (UAA) was similar to that of Huh-7 cells containing FK-E2 (UAA) (Fig. 3,B, compare lane 5 with lane 6). In other words, inhibition of PARP cleavage was also observed in cells containing FK-OE2 (UAA) (Fig. 3,B; compare lanes 5 and 6 with lane 4), indicating that E2 protein blocked TRAIL-induced apoptosis independent of core and E1. Antiapoptotic activity of E2 was also investigated by establishing a stable Huh-7 cell line expressing E2 and the signal sequence at the C-terminal region of E1 (Fig. 3,C). Cleavage of PARP by TRAIL was greatly reduced by the expression of E2 (Fig. 3 C, compare lane 4 with lane 3). These results indicate that E2 itself has antiapoptotic activity.

FIGURE 3.

Anti-apoptotic effect of E2 protein. A, Schematic diagrams of replicon FK-OE2 (UAA) and plasmid pCDNA-E2. Replicon FK-OE2 (UAA) contains the encephalomyocarditis virus (EMCV) IRES element for translation of E2 and termination codons at the N-terminal part of core and the C-terminal end of E2. This configuration ensures expression of E2 but not other structural proteins such as core and E1. Plasmid pCDNA-E2 contains the hygromycin selection marker, the C-terminal part of E1 (the signal sequence for E2), and E2 under the control of a CMV promoter. B, Cleavage of PARP in cells containing replicons FK-OE2 (UAA) (lanes 2 and 5) and FK-E2 (UAA) (lanes 3 and 6) was monitored by Western blot analysis before (lanes 1–3) and after (lanes 4–6) TRAIL treatment. C, Huh-7 cells were transfected with plasmid pCDNA-E2 (lanes 2 and 4) or control vector plasmid (lanes 1 and 3) and then selected with hygromycin B. The levels of E2 in the established Huh-7 cells were monitored by Western blot analysis (α-E2) before (lanes 1 and 2) and after (lanes 3 and 4) TRAIL treatment. Cleavage of PARP in control Huh-7 cells (lanes 1 and 3) and Huh-7 cells expressing E2 (lanes 3 and 6) was monitored by Western blot analysis (α-PARP) before (lanes 1 and 2) and after (lanes 3 and 4) TRAIL treatment.

FIGURE 3.

Anti-apoptotic effect of E2 protein. A, Schematic diagrams of replicon FK-OE2 (UAA) and plasmid pCDNA-E2. Replicon FK-OE2 (UAA) contains the encephalomyocarditis virus (EMCV) IRES element for translation of E2 and termination codons at the N-terminal part of core and the C-terminal end of E2. This configuration ensures expression of E2 but not other structural proteins such as core and E1. Plasmid pCDNA-E2 contains the hygromycin selection marker, the C-terminal part of E1 (the signal sequence for E2), and E2 under the control of a CMV promoter. B, Cleavage of PARP in cells containing replicons FK-OE2 (UAA) (lanes 2 and 5) and FK-E2 (UAA) (lanes 3 and 6) was monitored by Western blot analysis before (lanes 1–3) and after (lanes 4–6) TRAIL treatment. C, Huh-7 cells were transfected with plasmid pCDNA-E2 (lanes 2 and 4) or control vector plasmid (lanes 1 and 3) and then selected with hygromycin B. The levels of E2 in the established Huh-7 cells were monitored by Western blot analysis (α-E2) before (lanes 1 and 2) and after (lanes 3 and 4) TRAIL treatment. Cleavage of PARP in control Huh-7 cells (lanes 1 and 3) and Huh-7 cells expressing E2 (lanes 3 and 6) was monitored by Western blot analysis (α-PARP) before (lanes 1 and 2) and after (lanes 3 and 4) TRAIL treatment.

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TRAIL-mediated apoptosis commences with the interaction of TRAIL with the TRAIL receptors, DR4 and DR5, on the surface of target cells (45, 46). To further explore the effects of E2 on TRAIL-mediated apoptosis, we investigated TRAIL-induced intracellular events in Huh-7 cells containing the various replicons. The levels of total TRAIL receptor protein in Huh-7 cells were the same regardless of the presence or absence of any tested replicon as shown by FACS analysis (data not shown). Interestingly, the levels of DR4 and DR5 on the surface of Huh-7 cells were dramatically reduced in replicon-containing cells. However, no difference in the levels of DR4 and DR5 on the cell surface was observed among the different replicon-containing cells (data not shown), suggesting that one or more nonstructural proteins blocked translocation of DR4 and DR5 to the cell surface. As there was no observable difference in DR4 and DR5 display on cells expressing the various replicons, and TRAIL treatment successfully induced apoptosis in the Huh-7 cells with reduced levels of surface-presented TRAIL receptors, it is unlikely that reduction of DR4 and DR5 was responsible for the inhibition of apoptosis by E2.

Exposure of cells to TRAIL triggers type II apoptosis in Huh-7 cells (15) through the activation of caspase-8, cleavage of Bid, loss of mitochondrial membrane potential, and release of cytochrome c from mitochondria. Therefore, we investigated whether E2 protein affects the levels of the Bcl-2 family proteins (Bcl-2 and Bcl-xL) responsible for decreasing the mitochondrial release of cytochrome c (47, 48). The total protein levels of these proteins in Huh-7 cells containing different replicons were the same before and after TRAIL treatment, as shown in Fig. 4. This indicates that changes in the amounts of proteins Bcl-2 and Bcl-xL may not be involved in E2-mediated inhibition of apoptosis (Fig. 4). However, large differences in the levels of cytosolic cytochrome c were detected between E2-expressing cells (FK-E2 (UAA), FK-p7 (UAA), and FK) and E2-null cells (Huh-7, FK-C399T+2, FK-core (UAA), and FK-E1 (UAA)) before and after TRAIL treatment (200 ng/ml) (Fig. 5). Note that higher amounts of cytosolic cytochrome c were observed in cells containing replicons FK-C399T+2, FK-core (UAA), and FK-E1 (UAA) compared with control Huh-7 cells before TRAIL treatment (Fig. 5,A). This finding may be related to the TRAIL hypersensitivity of cells containing replicons FK-C399T+2, FK-core (UAA), and FK-E1 (UAA) (Fig. 2,C). Low levels of cytosolic cytochrome c were detected in cells containing E2-expressing replicons FK-E2 (UAA), FK-p7 (UAA), and FK before TRAIL treatment (Fig. 5,A). Increased levels of cytosolic cytochrome c were detected in all cells when TRAIL was applied to the media (Fig. 5), although higher levels of cytosolic cytochrome c were detected in cells without E2 as compared with cells expressing E2 (Fig. 5,B, percentage). It is noteworthy that the ratios of cytosolic cytochrome c after and before TRAIL treatment differ in E2-null and E2-expressing cells. The levels of cytosolic cytochrome c was increased ∼1.6-fold in E2-null cells, but those were increased ∼1.2-fold in E2-expressing cells (Fig. 5,B, compare lanes 8–11 with lanes 12–14 in B/A). The data indicate that E2 not only reduces basal levels of cytosolic cytochrome c in cells before TRAIL treatment but also blocks release of cytochrome c after TRAIL treatment. These phenomena correlated well with caspase-3 activity in the TRAIL-treated cells (Fig. 2 D), and may suggest that E2 blocks apoptosis by inhibiting the release of cytochrome c from mitochondria. However, the molecular basis for this inhibition of cytochrome c release remains to be elucidated.

FIGURE 4.

Levels of Bcl-2 family proteins in replicon-containing cells. A, Levels of Bcl-xL, Bcl-2, and control actin proteins in Huh-7 cells before TRAIL treatment were examined by Western blot analysis. B, Levels of Bcl-xL, Bcl-2, and control actin proteins in Huh-7 cells after TRAIL treatment were examined by Western blot analysis. Cells were treated with 200 ng/ml TRAIL for 2 h. Western blot analysis was performed with polyclonal Abs against Bcl-xL and Bcl-2 and a mAb against human actin. The transfected replicons (top) are shown.

FIGURE 4.

Levels of Bcl-2 family proteins in replicon-containing cells. A, Levels of Bcl-xL, Bcl-2, and control actin proteins in Huh-7 cells before TRAIL treatment were examined by Western blot analysis. B, Levels of Bcl-xL, Bcl-2, and control actin proteins in Huh-7 cells after TRAIL treatment were examined by Western blot analysis. Cells were treated with 200 ng/ml TRAIL for 2 h. Western blot analysis was performed with polyclonal Abs against Bcl-xL and Bcl-2 and a mAb against human actin. The transfected replicons (top) are shown.

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FIGURE 5.

The effects of HCV proteins on the mitochondrial release of cytochrome c. A and B, Levels of cytochrome c were monitored by Western blot analysis. The levels of cytochrome c in total cell extracts (top) and cytosolic fractions (bottom) are shown. Levels of cytochrome c in Huh-7 cells were monitored before and after TRAIL treatment. Cells were treated with 200 ng/ml TRAIL for 2 h. Western blot analysis was performed with a mAb against cytochrome c. The relative amounts of cytosolic cytochrome c in the cells containing various replicons before (A) and after (B) TRAIL treatments are shown as a percentile (%), considering the amount of cytochrome c in Huh-7 cells before TRAIL treatment as 100%. Relative ratios (B/A) of cytosolic cytochrome c after and before TRAIL treatment (B/A) in each replicon-containing cells.

FIGURE 5.

The effects of HCV proteins on the mitochondrial release of cytochrome c. A and B, Levels of cytochrome c were monitored by Western blot analysis. The levels of cytochrome c in total cell extracts (top) and cytosolic fractions (bottom) are shown. Levels of cytochrome c in Huh-7 cells were monitored before and after TRAIL treatment. Cells were treated with 200 ng/ml TRAIL for 2 h. Western blot analysis was performed with a mAb against cytochrome c. The relative amounts of cytosolic cytochrome c in the cells containing various replicons before (A) and after (B) TRAIL treatments are shown as a percentile (%), considering the amount of cytochrome c in Huh-7 cells before TRAIL treatment as 100%. Relative ratios (B/A) of cytosolic cytochrome c after and before TRAIL treatment (B/A) in each replicon-containing cells.

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The effect of E2 on survival of Huh-7 cells was assessed by measuring the colony forming efficiencies of cells containing the different replicon RNAs. The effect of RNA size on colony formation was minimal because the various RNA constructs were designed to be functionally similar in size (Fig. 1,A). Huh-7 cells were transfected with the various replicons, and colonies containing replicon RNAs were selected by cultivating the cells in the presence of G418 for 15 days before staining with crystal violet (Fig. 6). Cells expressing the E2-null replicons FK-C399T+2, FK-core (UAA), and FK-E1 (UAA) formed ∼60 colonies each. In contrast, cells containing the E2-expressing replicons FK-E2 (UAA), FK-p7 (UAA), and FK produced ∼1500 colonies each (Fig. 6). Moreover, replicon FK-OE2 (UAA), which expressed E2 but not core, E1, p7, or NS2, showed similar colony forming efficiency to replicon FK-E2 (UAA) (data not shown). These results strongly suggest that E2 augments proliferation of HCV replicons and/or inhibits cell death of host Huh-7 cells.

Studies on host-HCV interactions had been hampered by the lack of an efficient cell culture system until the recent development of a highly infectious HCV clone and a new cell line supporting cultivation of the virus (49, 50, 51). Previously, researchers have investigated the roles of HCV structural proteins using chimeric viruses in which the structural genes of Yellow Fever virus (YFV) or Semliki Forest virus (SFV) were replaced by the structural genes of HCV (52, 53). Other studies have used ectopic expression of HCV proteins in cell lines and transgenic mice to investigate the roles of viral proteins (52, 53, 54). The HCV replicon mimicking a part of the natural infection cycle is an alternative model system that could be used in investigation of functions of viral proteins. In this study, we generated replicons expressing nonstructural proteins essential for viral RNA replication along with different structural and nonstructural proteins by introducing artificial stop codons (Fig. 1 A). We used this system to investigate the effects of various viral proteins that are not essential for viral RNA replication.

Comparative analysis of the effects of various HCV replicons on Huh-7 cells revealed that E2 expression inhibited TRAIL and etoposide-induced apoptosis. To investigate the molecular basis of the antiapoptotic function of E2, we monitored the levels of TRAIL receptors DR4 and DR5, as well as antiapoptotic proteins Bcl-2 and Bcl-xL. The levels of DR4 and DR5 remained the same regardless of the presence or absence of the various replicons in Huh-7 cells. Curiously, the level of DR4 and DR5 on the surface of cells was reduced by the presence of all replicons (data not shown), regardless of the expression level of E2. These results indicate that one or more nonstructural protein blocked transport of DR4 and DR5 to the cell surface. Recently, it was reported that NS4A/B interferes with protein trafficking, leading to reduced Ag presentation mediated by MHC class I molecules (55). Thus, it is possible that NS4A/B, which was expressed in all of our tested replicon-containing cells, may have blocked translocation of the TRAIL receptors. Further work will be necessary to confirm this activity of NS4A/B and investigate physiological implications of the cell surface reduction of TRAIL receptors. It should be noted that the amount DR4 and/or DR5 on the surface of replicon-containing Huh-7 cells was sufficient for TRAIL-induced apoptosis under our experimental conditions (Figs. 2 and 3). The total protein levels of Bcl-2 and Bcl-xL, which block cytochrome c release from mitochondria, were the same regardless of the presence or absence of the replicons and E2 protein (Fig. 4).

However, there were differences in cytosolic cytochrome c levels among the various replicons-containing cells. The level of cytosolic cytochrome c in E2-expressing cells was lower than levls in E2-null cells before and after TRAIL treatment (Fig. 5). Increased levels of cytosolic cytochrome c in control Huh-7 cells and Huh-7 cells containing the E2-null replicons FK-C399T+2, FK-core (UAA) and FK-E1 (UAA) was detected in TRAIL-treated cells (Fig. 5,B, lanes 8–11). This increased level of cytosolic cytochrome c seemed to be sufficient to trigger activation of downstream caspases such as caspases 9, 3, and 7. In contrast, Huh-7 cells containing replicons expressing E2 (FK-E2 (UAA), FK-p7 (UAA), and FK) showed lower levels of cytosolic cytochrome c even after TRAIL treatment (Fig. 5,B, lanes 12–14). This decreased level of cytosolic cytochrome c in the E2-expressing cells upon TRAIL treatment was correlated well with antiapoptotic activity of E2-producing cells (Fig. 2 B, lanes 6–8).

As an attempt to understand the molecular basis of the antiapoptotic effect of E2, we observed the cleavage of Bid after TRAIL treatment. Interestingly reduced levels of Bid cleavage were observed in the E2-expressing cells after TRAIL treatment (data not shown). However, we do not know whether the reduced cleavage of Bid results in blockage of cytochrome c release from the mitochondria, or the blockage of cytochrome c release results in the reduced cleavage of Bid because activation of apoptosis via mitochondria forms a positive amplification loop through the cleavage of caspase-8 by the activated caspase-3. In other words, activated caspase-3 cleaves caspase-8, and the activated caspase-8 in turn cleaves of Bid. The cleaved Bid may further promote mitochondrial dysfunctions in a positive amplification loop by triggering cytochrome c release (56).

Curiously, higher levels of TRAIL-induced apoptosis were detected in Huh-7 cells containing the E2-null replicons FK-C399T+2, FK-core (UAA), and FK-E1 (UAA) as compared with control Huh-7 cells (Fig. 2). This seems to be related with elevated level of cytosolic cytochrome c in these cells before the treatment of TRAIL compared with control Huh-7 cells (Fig. 5,A). The apoptotic sensitivity of replicon FK-C399T+2-containing cells may be attributed to viral proteins essential for viral RNA replication (34, 35) and/or to viral RNAs possibly through IFN signaling. Slightly increased levels of caspase-3 activity were detected in cells containing FK-core (UAA) and FK-E1 (UAA), as compared with cells containing FK-C399T+2 (Fig. 2,D; compare lanes 9 and 11 with lanes 15, 17, 21, and 23). This result was likely due to apoptotic hypersensitivity caused by core protein, as previously described (57, 58). In this regard, it is worthy to note that core was shown to enhance apoptotic effect of TRAIL by augmenting release of cytochrome c into cytosol (33). The antiapoptotic effect of E2 was strong enough to nullify the sensitization of apoptosis by other viral proteins (Fig. 2C, compare lanes 3–5 with lanes 6–8). Moreover, Huh-7 cells containing replicons expressing E2 were even less sensitive than control Huh-7 cells to TRAIL (Fig. 2 C, compare lane 2 with lane 6). Taken together, these results suggest that blockage of apoptosis of HCV-infected cells by E2 likely plays a crucial role in maintaining persistent infection status.

Several lines of evidence presented in this study suggest that E2 blocks apoptosis induced by various signaling pathways. First, E2 blocked apoptosis induced by TRAIL (Fig. 2) and by combined treatment with TNF and cycloheximide (data not shown). Furthermore, E2 inhibited cell death induced by etoposide that triggers mitochondria-mediated apoptosis (Fig. 2,F). Second, E2 blocked cell death, as shown by increased colony forming efficiencies in cells containing E2-expressing replicons (Fig. 6). Third, E2 blocked mitochondrial release of cytochrome c, which is one of the key steps in the type II pathway of apoptotic induction (Fig. 5 B). In this respect, it is noteworthy that a transgenic mouse expressing the HCV core, E1, E2, p7, and NS proteins was resistant to apoptosis induced by an anti-Fas Ab (54). We speculate that the E2 protein expressed in this mouse model contributed to the blockage of Fas-induced apoptosis. Moreover, it was shown very recently that a HCV protein in a full-length replicon provided resistance against cell death induced by serum starvation even though identity of the protein responsible for the antiapoptotic effect was not known (59). We speculate that the antiapoptotic effect may be attributed to E2.

Several reports have suggested that E2 blocks the activities of dsRNA-activated PKR and PKR-like endoplasmic reticulum-resident kinase that inhibit translation through phosphorylation of eukaryotic initiation factor (eIF)-2α subunit (37, 60, 61). Accordingly, we investigated whether E2 nullifies apoptosis through the inactivation of PKR and/or of PKR-like endoplasmic reticulum-resident kinase by measuring the phosphorylation level of the eIF-2α subunit, which is a substrate of both PKR and PKR-like endoplasmic reticulum-resident kinase. No change in phosphorylation level of eIF-2α was observed in Huh-7 cells regardless of the presence or absence of replicons and E2 protein (data not shown), suggesting that inhibition of eIF-2α phosphorylation may not be the major apoptosis-blocking activity of E2 protein in replicon containing cells.

Many viruses encode proteins that suppress or delay apoptosis of host cells long enough for the virus to replicate or establish persistent infection (62, 63, 64). Such “death-preventing” viral genes may also contribute to cancer generation (particularly in terms of hepatocellular carcinoma), as they may prevent death triggered by internal signaling cascades or external stimuli. Therefore, investigation of the molecular basis of E2 function in antiapoptosis will add to our understanding of persistent HCV infection and tumorigenesis.

We are grateful to Chang-Yuil Kang (College of Pharmacy, Seoul National University) for E2 Ab, and Yong-Keun Jung (Department of Life Science, Kwangju Institute of Science and Technology) for caspase-9 Ab.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by Grant Molecular Cellular BioDiscovery Research Group (M10501000022-05-N0100-02200) from the Ministry of Science and Technology (Korea), Grants 02-PJ2-PG1-CH16-0002 and 0405-VN02-0702-0008 from Korea Health Industry Development Institute, Grant Systems-BioDynamics-National Core Research Center (R15-2004-033-01001-0) from the Korea Research Foundation, Grant FPR05B 1-310 of the 21C Frontier Functional Proteomics Center project, Korean Ministry of Science and Technology, and a grant from POSCO.

3

Abbreviations used in this paper: HCV, hepatitis C virus; PARP, poly(ADP-ribose) polymerase; NS, nonstructural; PKR, protein kinase R; AMC, 7-amino-4-methylcoumarin; IRES, internal ribosomal entry site; eIF, eukaryotic initiation factor.

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