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under oxidative stress
1 Faculty of Life Sciences, The University of Manchester, Jackson's Mill, PO Box 88, Sackville Street, Manchester M60 1QD, UK
2 Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK
3 Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK
Correspondence
Shiu-Wan Chan
shiu-wan.chan{at}manchester.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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phosphorylation. A phosphomimetic of eIF2, which mimics the structure of the phosphorylated eIF2, was sufficient to repress translation in the absence of H2O2. In H2O2-resistant HepG2 cells, H2O2 activated both HCV IRES-mediated and cap-dependent translation, associated with an increased level of phospho-eIF2. It was postulated that H2O2 might stimulate translation in HepG2 cells via an eIF2-independent mechanism, whereas the simultaneous phosphorylation of eIF2 repressed part of the translational activities. Indeed, the translational repression was released in the presence of a non-phosphorylatable mutant, eIF2-SA, resulting in further enhancement of both translational activities after exposure to H2O2. In HuH7 cells, which exhibited an intermediate level of sensitivity towards H2O2, both HCV IRES-mediated and cap-dependent translational activities were upregulated after treatment with various doses of H2O2, but the highest level of induction was achieved with a low level of H2O2, which may represent the physiological level of H2O2. At this level, the HCV IRES-mediated translation was preferentially upregulated compared with cap-dependent translation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus (Kato et al., 1990). Its genome encodes a single polyprotein of 
3010 aa, which is processed into structural (core, envelope glycoproteins E1 and E2, p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins (Moradpour et al., 2002).
In contrast to cap-dependent translation of the majority of cellular mRNAs, the initiation of HCV translation is cap-independent and mediated by a highly conserved internal ribosome entry site (IRES) element located within the 5' non-coding terminal region (NTR) and extends into the core protein open reading frame (Reynolds et al., 1995; Tsukiyama-Kohara et al., 1992). The involvement of the X region in the 3' NTR and viral proteins in the modulation of HCV IRES-mediated translation has also been implicated (Boni et al., 2005; Ito et al., 1998; Kalliampakou et al., 2005). Both canonical and non-canonical eukaryotic initiation factors (eIFs) have been shown to play essential roles in IRES-dependent translation. The HCV IRES can directly recruit the 40S ribosomal subunit before forming an initiation complex with eIF2 and eIF3 (Pestova et al., 1998). eIF2B
and eIF2 have also been identified as co-factors in HCV IRES-mediated translation (Krüger et al., 2000). Several cellular RNA-binding proteins, including La autoantigen and polypyrimidine tract-binding protein, have been implicated in efficient HCV IRES-mediated translation (Ali & Siddiqui, 1995, 1997).
Accumulation of reactive oxygen species (ROS) and the generation of oxidative stress have been implicated in the development of a number of inflammatory diseases, including viral hepatitis (Schwarz, 1996). ROS can cause oxidative damage to intracellular macromolecules and modulate cellular signal transduction pathways. Immune recognition of infected hepatocytes triggers the release of ROS (e.g. superoxide anion, hydrogen peroxide) from sequestered phagocytes and activated macrophages. It is evident that oxidative stress is associated with HCV infection. Chronic hepatitis C patients present elevated blood and hepatic levels of ROS, with increased lipid peroxidation and decreased hepatic glutathione (De Maria et al., 1996; Paradis et al., 1997). Intrahepatic gene expression profiling using microarray analysis has demonstrated upregulation of the oxidative stress-inducible genes in hepatitis C samples (Yamashita et al., 2001). Data from studies in vitro and in vivo suggest the direct involvement of HCV replication and gene expression in the generation of ROS. Replication of the subgenomic replicon of HCV in neomycin-selected cultured cells results in increased oxidative stress (Qadri et al., 2004). HCV core, NS3 and NS5A proteins are capable of inducing oxidative stress in cultured hepatocytes, monocytes and isolated mitochondria, and transgenic mice carrying the structural proteins exhibit elevated levels of ROS and are more susceptible to oxidant injury (Bureau et al., 2001; Gong et al., 2001; Korenaga et al., 2005; Moriya et al., 2001; Okuda et al., 2002). In the case of NS5A, it has been proposed that oxidative stress is triggered by the increased efflux of Ca2+ from the endoplasmic reticulum as a result of endoplasmic reticulum stress (Gong et al., 2001).
It is unclear how the virus itself can circumvent oxidative stress in terms of replication, translation and survival. To understand how virus translation is modulated under oxidative stress, we compared HCV IRES-mediated and cap-dependent translational activities using a bicistronic reporter construct (Collier et al., 1998).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-SA were obtained from Costas Koumenis (Koumenis et al., 2002) and maintained in Ham's F12 medium. All media were supplemented with 10 % fetal calf serum, 100 U penicillin ml–1, 100 µg streptomycin ml–1 and 2 mM glutamate.
XTT viability assay.
An XTT (sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) assay was performed according to the manufacturer's instructions (Cell Proliferation kit II; Roche). Cells seeded in 96-well plates were treated with H2O2 for 24 h before the addition of XTT. Readings were taken at 450 nm using a 650 nm reference filter and were corrected for background absorbance. The values shown represent means±SEM of three independent experiments performed in triplicate and were expressed relative to the untreated control, which was set as 1 and represented 100 % viability.
ROS measurement.
The generation of intracellular ROS was measured using the probe 2',7'-dichlorofluorescein diacetate (DCFH-DA; Sigma) (Wang & Joseph, 1999). Cells seeded in 96-well plates were washed with PBS and pre-loaded with freshly prepared 100 µM DCFH-DA for 30 min at 37 °C. Following several PBS washes, cells were exposed to 100 µl medium containing serial dilutions of H2O2. The fluorescence from each well was measured immediately using a microplate fluorimeter (Twinkle; Berthold Technologies) with the excitation filter set at 485 nm and the emission filter at 535 nm with the temperature maintained at 37 °C. Data were collected every 5 min for 90 min. Each data point represents the mean±SEM of three independent experiments performed in triplicate.
Plasmids.
The bicistronic construct pRL encoding the Renilla luciferase (RLuc) and firefly luciferase (FLuc) genes under the control of a T7 promoter and a genotype 1b HCV IRES has been described previously (Collier et al., 1998). In this study, the bicistronic construct was subcloned to generate the plasmid pRFHCV1b, which contained the RLuc gene under the control of the cytomegalovirus (CMV) promoter and the FLuc gene under the control of the HCV IRES (see Fig. 1c). Briefly, a SacI–HindIII fragment containing the bicistronic construct was excised from pRL and subcloned into the vector pEGFP-N1 (Clontech). From this, a XhoI–ApaI fragment was excised and subcloned into the vector pcDNA3.1 (Invitrogen) to generate pRFHCV1b. Translation from the RLuc gene was cap-dependent, whereas translation from the FLuc gene was IRES-mediated. A promoterless (
CMV) pRFHCV1b plasmid was constructed by excising an MluI–XhoI fragment containing the CMV promoter from pRFHCV1b. Plasmids encoding eIF2, eIF2-SA, eIF2-SD and the empty vector control hCD2 were obtained from David Ron (Novoa et al., 2001).
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Dual luciferase assay.
The activities of RLuc and FLuc were measured in relative light units over 10 s with a luminometer (Lumat LB9507; Berthold Technologies) using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Protein concentrations were determined using the Bradford assay or an RC-DC protein assay kit (Bio-Rad). Luciferase activities, normalized with respect to total protein, were expressed relative to the untreated or empty vector controls. The IRES/cap ratio represented the ratio of FLuc to RLuc activity and was expressed relative to the control, which was set as 1. The values obtained represented means±SEM of three independent experiments performed in triplicate.
RT-PCR.
Total RNA from each well was extracted into 200 µl TRIzol (Invitrogen) or RNA-Bee (Tel-Test) according to the manufacturers' instructions. We confirmed that RNA extracted by this method was free from contaminating DNA by the absence of PCR amplification from DNase-free RNase I-digested RNA samples. Nevertheless, to ensure that the fragments detected were not amplified from residual vector DNA, the RNA sample was pre-treated with 1 U RNase-free DNase I (Roche). Total RNA (1 µg) was reverse transcribed and amplified by multiplexed or separate RT-PCR using the Titan One Tube RT-PCR System (Roche) with the following primer pairs: FLuc: 5'-CTGAAGGGATCGTAAAAACAGC-3' and 5'-GATTACCAGGGATTTCAGTCG-3'; RLuc: 5'-CCACATATTGAGCCAGTAGC-3' and 5'-CCATGATAATGTTGGACGAC-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-CCTGTTCGACAGTCAGCCG-3' and 5'-CGACCAAATCCGTTGACTCC-3'. The reverse transcription step was performed at 50 °C for 30 min, followed by 2 min of denaturation at 94 °C and 35 cycles of PCR using the following conditions: 94 °C for 10 s, 60 or 50 °C for 30 s and 68 °C for 45 s or 2 min, with a final extension at 68 °C for 5 min. The intensity of the luciferase bands was measured using IMAGEQUANT 5.0, normalized against an internal control (GAPDH) and expressed relative to the 0 µM H2O2-treated controls, which were set as 1.
Western blotting.
Cells treated with H2O2 were harvested into 1x SDS-PAGE sample buffer. Protein concentrations were measured using the Bradford assay or an RC-DC protein assay kit. Western blotting was performed as described previously (Chan & Egan, 2005) using antibody specific for phospho-eIF2 (Cell Signalling), diluted 1 : 1000. Blots were stripped and reprobed with antibody recognizing total eIF2 (BioSource or Cell Signaling), diluted 1 : 1000. The intensity of the bands was measured with IMAGEQUANT 5.0, normalized against total eIF2 and expressed relative to the 0 h control, which was set as 1.
In vitro transcription.
In vitro transcripts were generated from the T7 promoter of pRL (Collier et al., 1998) or pRFHCV1b (this study) using mMESSAGE mMACHINE and MEGAscript transcription kits (Ambion) according to the manufacturer's instructions.
Statistical analyses.
Statistical analyses were performed using analysis of variance. A P value of less than 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A physiological level of H2O2 is important for signalling, whereas a cytotoxic level of H2O2 can be considered as oxidative stress. HCV infection is frequently associated with oxidative stress, and apoptosis and necrosis are common features of HCV-infected liver (De Maria et al., 1996; Dhillon & Dusheiko, 1995; Paradis et al., 1997). Therefore, we sought to study the translational responses of HCV under conditions of oxidative stress that induced apoptosis or necrosis. The range of H2O2 concentrations that induced different cellular responses in HeLa cells were established using XTT assays and phase-contrast microscopy (Fig. 1a, d
). HeLa cells remained viable until the concentration of H2O2 reached 10 µM. Concentrations greater than 10 µM were cytotoxic. The concentrations of H2O2 that induced cell death correlated with intracellular elevation of ROS, as measured by a fluorometric assay employing DCFH-DA (Fig. 1b). Concentrations of H2O2 that did not affect cell viability (<10 µM) did not increase intracellular ROS levels over that of the untreated control. To test the effect of oxidative stress on HCV IRES-mediated translation, HeLa cells were transiently transfected with the bicistronic construct pRFHCV1b (Fig. 1c) and then treated with concentrations of H2O2 that have been shown to generate intracellular ROS and to elicit different growth responses in HeLa cells (Fig. 1e). Bicistronic reporter vectors are now commonly used to study IRES-mediated translation relative to cap-dependent translation (Hellen & Sarnow, 2001). After 4 h of treatment, a low level of H2O2 (5 µM) that had little or no effect on cell viability also failed to affect translational activities significantly. In contrast, repression of both HCV IRES-mediated and cap-dependent translation was observed at apoptotic (50 µM) and necrotic (500 µM) levels of H2O2. At 50 µM H2O2, both HCV IRES-mediated and cap-dependent translation were reduced to a similar extent, resulting in an unchanged IRES/cap ratio. However, treatment with 500 µM H2O2 caused considerably greater reduction in cap-dependent translation than HCV IRES-mediated translation, resulting in an overall fourfold increase in the IRES/cap ratio. These results suggested that initiation of translation from the HCV IRES was less sensitive or more resistant than cap-dependent translation to severe stress conditions. Using semi-quantitative RT-PCR, equal amounts of transcripts were amplified from each transfection reaction, confirming that changes in the IRES activity were translational and not transcriptional (Fig. 1f). For further confirmation of the effects of H2O2 on translational activities and to exclude the possibility that the effects obtained were derived from a cryptic promoter within the IRES sequence (Dumas et al., 2003), we repeated the experiments using RNA transfection with the pRL transcript (Collier et al., 1998) and obtained similar responses in translational activities after treatment with H2O2 (Fig. 1g).
FLuc activity does not derive from a cryptic promoter or spliced transcripts
The existence of cryptic promoters within IRES sequences has been reported in several cellular IRESs and the HCV IRES (Dumas et al., 2003; Han & Zhang, 2002). As we used DNA transfection in some of our experiments, it could be argued that the effects obtained were derived from a cryptic promoter. For example, the difference in band intensity of the RLuc and FLuc fragments in Fig. 1(f) could be due to the production of additional FLuc transcripts from a cryptic promoter sequence within the HCV IRES. However, it was equally possible that it merely reflected the difference in PCR efficiency of the two sets of primers. Supporting the latter possibility was the similar degree of differential amplification of the RLuc and FLuc fragments from an in vitro transcript generated from the T7 promoter of the bicistronic vector pRFHCV1b and from transfected cells (Fig. 2a). To exclude the possibility of the presence of a cryptic promoter within the HCV IRES sequence, we compared the RLuc and FLuc activities in cells transfected with the bicistronic construct with and without the CMV promoter (Fig. 2b). Removal of the CMV promoter abolished the CMV-driven RLuc activity and, at the same time, FLuc activity, suggesting the absence of a cryptic promoter in our bicistronic construct pRFHCV1b, in at least three of the cell lines (HeLa, HepG2 and A549) used in this study. Also, in agreement with results from other studies (Sherrill et al., 2004; Van Eden et al., 2004a, b), we did not detect any aberrantly spliced transcripts in HeLa and HepG2 cells following transfection with the bicistronic DNA, as only an RT-PCR fragment of the expected size of 1.93 kb was amplified from the outer primers (Fig. 2c).
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phosphorylation in a time- and dose-dependent manner (O'Loghlen et al., 2003). When we examined H2O2-treated HeLa cells, we also revealed a time- and dose-dependent phosphorylation of eIF2 in these cells (data not shown). Therefore, we investigated the role of phospho-eIF2 in modulating both HCV IRES-mediated and cap-dependent translation under oxidative stress. Indeed, the kinetics of eIF2 phosphorylation correlated with the kinetics of translational repression observed in HeLa cells after treatment with H2O2 (Fig. 3a, b). The degree of eIF2 phosphorylation remained unaffected following treatment with sub-apoptotic levels of H2O2 (5 and 10 µM). This was reflected in the absence of change in translational activities observed at these concentrations. However, increased phosphorylation of eIF2 was detected at apoptotic and necrotic levels of H2O2 (50 and 500 µM, respectively) and these concentrations also repressed translational activities.
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represses HCV IRES-mediated and cap-dependent translation in the absence of oxidative stress phosphorylation in modulating translational activities, we transfected HeLa cells with the eIF2 mutants eIF2-SA and eIF2-SD (Fig. 3c). As a control, cells were transfected with wild-type eIF2, which resulted in a slight elevation in translational activities. Phosphorylation of eIF2 occurs at serine 51 (Choi et al., 1992). Replacement of serine with alanine results in a non-phosphorylatable eIF2, eIF2-SA, which acts as a dominant-negative mutant by exchanging with endogenous eIF2 in the ternary complexes (Choi et al., 1992). Transfection of cells with the non-phosphorylatable eIF2-SA resulted in further translational activation compared with transfection with wild-type eIF2. Conversely, in the phosphomimetic eIF2-SD, serine 51 has been replaced with aspartate to mimic the structure of phosphorylated eIF2 (Choi et al., 1992). In this case, even without stimulation with H2O2, transfection of HeLa cells with eIF2-SD alone was sufficient to repress both HCV IRES-mediated and cap-dependent translation, suggesting that phosphorylation of eIF2 plays a modulating role in both HCV IRES-mediated and cap-dependent translation.
H2O2 activates HCV IRES-mediated and cap-dependent translation in HepG2 cells
HCV is a hepatotropic virus and therefore we examined the translational responses in a hepatocyte cell line, HepG2. We chose HepG2 cells because they are highly differentiated hepatocytes and physiologically resemble primary hepatocytes more closely; thus, HepG2 is a good cell model in studies of HCV, which infects differentiated hepatocytes. HepG2 cells were more resistant to H2O2 and required 500 µM exogenously added H2O2 and a correspondingly greater increase in the intracellular level of ROS to induce mild apoptosis (Fig. 4a, b). Translational activities were not affected by low concentrations of H2O2, such as 20 µM, that did not affect intracellular ROS levels or cell viability (Fig. 4c). Translation was activated by higher concentrations of H2O2, such as 200 µM, which also caused a sharp rise in intracellular ROS levels but which did not affect cell viability or transcriptional activity (Fig. 4c, d). We confirmed that the changes in IRES activity were translational and not transcriptional by the amplification of equal amounts of transcripts from each transfection reaction using semi-quantitative RT-PCR (Fig. 4d) and also by using RNA transfection (data not shown). At 2 mM H2O2, both luciferase and transcriptional activities decreased, probably as a result of cell toxicity.
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). A 30 min transient exposure of HepG2 cells to levels of H2O2 up to 2 mM did not affect viability, but was sufficient to induce transient elevation in intracellular ROS levels and, in turn, activated IRES- and cap-dependent luciferase activities in a dose-dependent manner. We confirmed that changes in the IRES activity were translational and not transcriptional by the amplification of equal amounts of transcript from each transfection reaction by using semi-quantitative RT-PCR (Fig. 5d).
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enhances translation after H2O2 treatment phosphorylation in HepG2 cells exposed to 200 µM H2O2, there was also an increase in the level of phospho-eIF2, which peaked at 2 h before returning to baseline level at 24 h post-treatment (Fig. 6a). As phospho-eIF2 repressed translation in HeLa cells in contrast to HepG2 cells where translational activities were increased after treatment with 200 µM H2O2, we reasoned that H2O2 might have stimulated translation in HepG2 cells via an eIF2-independent mechanism, whereas the simultaneous phosphorylation of eIF2 repressed part of the translational activities. Indeed, in HepG2 cells transfected with the non-phosphorylatable eIF2-SA mutant, further enhancement of both translational activities was observed after exposure to 200 µM H2O2, suggesting a role for the non-phosphorylatable eIF2-SA in releasing the translational repression (Fig. 6b). In contrast, HepG2 cells transfected with the phosphomimetic eIF2-SD failed to exhibit any translational upregulation in response to 200 µM H2O2.
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-SA mutant. The low level obtained could be due to the low efficiency of transient transfection. This is especially true with HepG2 cells, which have been known to be rather difficult to transfect. To address the role of phospho-eIF2 in translational repression further, we sought the use of a homogeneous population of cells stably expressing the non-phosphorylatable mutant eIF2-SA. As stable eIF2-SA hepatocyte cell lines are not available, we used a lung carcinoma cell line, A549, stably expressing eIF2-SA instead (Koumenis et al., 2002). This cell line has also been well characterized in that the eIF2-SA mutant cells fail to exhibit eIF2 phosphorylation under oxidative stress (Koumenis et al., 2002). First, we confirmed that wild-type A549 was similar to HepG2 in its sensitivity and translational responses to H2O2 treatment (Fig. 7). Wild-type A549 also exhibited a H2O2-resistant phenotype, although sensitivity was observed at concentrations >400 µM H2O2 (Fig. 7a). Similar to HepG2 cells, both HCV IRES-mediated and cap-dependent translation in wild-type A549 were upregulated following exposure to 200 µM H2O2 (Fig. 7b). We then examined the effect of stable expression of eIF2-SA on translation. The eIF2-SA mutant A549 was as resistant as wild-type A549 to H2O2 (Fig. 7a). In the eIF2-SA mutant cells, HCV IRES-mediated and cap-dependent translation was further enhanced upon H2O2 stimulation compared with wild-type A549 (Fig. 7b). These results also indicated that phospho-eIF2 plays a modulating role on translation in H2O2-resistant cells.
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), the hepatocyte cell line HuH7 has been employed widely in the study of HCV because it represents the cell line most supportive of HCV replication and even of a complete infectious cycle with the recently established cell-culture system (Wakita et al., 2005). Therefore, we sought to characterize the translational responses to H2O2 in HuH7 cells. The sensitivity of HuH7 cells to H2O2 was in between that of HepG2 and HeLa cells (Fig. 8a, b). H2O2 at 20 µM induced a slight elevation of intracellular levels of ROS, but cells remained as viable as the untreated control; thus, this concentration may represent a physiological level of H2O2. At 100 µM H2O2, a high level of cell death was observed with a modest elevation of intracellular levels of ROS. Thus, this concentration may represent a cytotoxic level of H2O2 and can be considered as oxidative stress. At 50 µM H2O2, a low (although insignificant) level of cell death was observed with a modest elevation of intracellular levels of ROS. Thus, this concentration may represent a critical dose of H2O2 functioning as a physiological/cytotoxic switch. These all have implications in HCV studies, because, for the duration of a chronic infection, the virus will have frequent exposure to different levels of H2O2, ranging from physiological to cytotoxic to pathological. Transfection of HuH7 cells with the bicistronic pRL IRES RNA transcript showed an inverse dose-dependent upregulation in both cap-dependent and HCV IRES-mediated translational activities (Fig. 8c). A physiological dose of 20 µM H2O2 induced the highest levels of translational activities, with less marked increases in translational activities at 50 µM H2O2, followed by 100 µM H2O2. Moreover, only at 20 µM H2O2 was the HCV IRES preferentially upregulated by twofold compared with cap-dependent translation. At 50 and 100 µM H2O2, both cap-dependent and HCV IRES-mediated translation responded similarly. Interestingly, elevation of the level of eIF2 phosphorylation was only obvious at 100 µM H2O2 where the level peaked at 30 min (Fig. 8d).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We demonstrated that, at increased concentrations of H2O2, the response varied according to the sensitivity of each cell type to H2O2, highlighting the spectrum of responses exhibited by different cell lines. The results in HepG2 and HuH7 cells, two more relevant cell lines in which to study HCV IRES activity, indicated that HCV IRES activity may also be influenced by cell type. HepG2 cells are very resistant to H2O2, perhaps reflecting a more robust anti-oxidant defence in these cells. At concentrations of H2O2 that increased levels of ROS but failed to induce a cell response that affected cell viability, HCV IRES activity was upregulated. By contrast, HuH7 and HeLa cells exhibited a more H2O2-sensitve phenotype and demonstrated a dose-dependent decrease in cell viability. Cell-type specificity was also observed in IRES response to HCV core protein expression (Li et al., 2003). This also underscores the importance of using relevant cell lines in such studies.
In H2O2-sensitive cells, such as HeLa cells, translation was repressed with H2O2 treatment, whereas in H2O2-resistant cells, such as HepG2 cells, translation was upregulated. Irrespective of whether translation was repressed or upregulated, both HCV IRES-mediated and cap-dependent translation responded in a similar way. This is in contrast to the regulation of many other viral and cellular IRESs. An essential step during productive picornaviral infection is the viral protease-mediated shut-off of host translation (Belsham & Sonenberg, 1996). The picornaviral IRES element enables virus translation to proceed whilst that of the host cell has been shut off. An increasing number of eukaryotic mRNAs, often encoding regulatory proteins and possessing an IRES within their 5' NTR, have recently been identified (Holcik et al., 2000). Some of these have been shown to function preferentially when cap-dependent translation is impaired under various stress conditions (Holcik et al., 2000). The HCV IRES differs in both length and structure from other IRESs and has a simpler requirement for translational factors (Beales et al., 2003; Pestova et al., 1998). Additionally, in contrast to picornaviruses, HCV establishes a chronic infection and, consequently, the role of the IRES may be functionally different to other viral elements. We speculate that it is necessary to confer translational competence to cellular genes in order to maintain a chronic infection; thus, HCV IRES-mediated and cap-dependent translation are regulated in a similar way under oxidative stress.
One exception to this rule of co-regulation of HCV IRES-mediated and cap-dependent translation is the preferential translation by the HCV IRES during exposure of HuH7 cells to a low (physiological) level of H2O2, suggesting that regulation of HCV IRES translation may be different at physiological and cytotoxic levels of H2O2. Moreover, it is tempting to speculate that molecules activated during physiological H2O2 signalling may be utilized to facilitate HCV IRES translation under such conditions.
Our results suggest that both HCV IRES-mediated and cap-dependent translation are modulated by phosphorylation of eIF2. Phospho-eIF2 negatively regulates global cellular cap-dependent translation; however, increasing evidence suggests that it is also a positive regulator of certain viral and cellular IRES elements (Fernandez et al., 2002a, b; Gerlitz et al., 2002). For example, under certain conditions of cellular stress when most translational activities are shut off, translation from IRES elements is differentially upregulated. This is selectively advantageous to the life cycle and survival of some viruses and is essential for the regulation of specific genes involved in cellular processes such as survival and differentiation (Fernandez et al., 2002a, b; Gerlitz et al., 2002). It is not known whether all IRES elements are positively regulated by phospho-eIF2. In this study, we have shown that the induction of oxidative stress repressed HCV IRES-mediated translation in HeLa cells. The observed reduction coincided with the increased phosphorylation status of eIF2, suggesting a negative regulatory role for phospho-eIF2 in HCV translation. This finding was further supported by phosphomimetic eIF2 studies in HeLa cells and the rescue of translational activities in HepG2 cells and in A549 cells expressing a non-phosphorylatable eIF2. In HuH7 cells, the reduction in the degree of translational activation at 100 µM H2O2 (compared with that at 20 µM H2O2) coincided with an elevation in the level of phospho-eIF2, although we cannot currently link the two events together in the absence of additional mechanistic studies. Negative regulation of viral IRES elements has been demonstrated previously. Increased HCV IRES activity has been observed with reduced levels of phospho-eIF2 in replicon cells (He et al., 2003). In contrast, RNA-activated protein kinase-induced HCV IRES translation seems to be dependent on phospho-eIF2 (Rivas-Estilla et al., 2002).
Together these results suggest strongly that not all IRES elements are subject to positive regulation by phospho-eIF2 and that HCV IRES activity may be dependent on the cell type and the type of stimuli.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Ali, N. & Siddiqui, A. (1997). The La antigen binds 5' noncoding region of the hepatitis C virus RNA in the context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation. Proc Natl Acad Sci U S A 94, 2249–2254.
Beales, L. P., Holzenburg, A. & Rowlands, D. J. (2003). Viral internal ribosome entry site structures segregate into two distinct morphologies. J Virol 77, 6574–6579.
Belsham, G. J. & Sonenberg, N. (1996). RNA–protein interactions in regulation of picornavirus RNA translation. Microbiol Rev 60, 499–511.
Boni, S., Lavergne, J.-P., Boulant, S. & Cahour, A. (2005). Hepatitis C virus core protein acts as a trans-modulating factor on internal translation initiation of the viral RNA. J Biol Chem 280, 17737–17748.
Bureau, C., Bernad, J., Chaouche, N., Orfila, C., Béraud, M., Gonindard, C., Alric, L., Vinel, J.-P. & Pipy, B. (2001). Nonstructural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase. J Biol Chem 276, 23077–23083.
Chan, S.-W. & Egan, P. A. (2005). Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response. FASEB J 19, 1510–1512.
Choi, S.-Y., Scherer, B. J., Schnier, J., Davies, M. V., Kaufman, R. J. & Hershey, J. W. B. (1992). Stimulation of protein synthesis in COS cells transfected with variants of the -subunit of initiation factor eIF-2. J Biol Chem 267, 286–293.
Choi, J., Lee, K. J., Zheng, Y., Yamaga, A. K., Lai, M. M. C. & Ou, J.-H. (2004). Reactive oxygen species suppress hepatitis C virus RNA replication in human hepatoma cells. Hepatology 39, 81–89.[CrossRef][Medline]
Collier, A. J., Tang, S. & Elliott, R. M. (1998). Translation efficiencies of the 5' untranslated region from representatives of the six major genotypes of hepatitis C virus using a novel bicistronic reporter assay system. J Gen Virol 79, 2359–2366.[Abstract]
Davies, K. J. A. (1999). The broad spectrum of responses to oxidants in proliferating cells: a new paradigm for oxidative stress. IUBMB Life 48, 41–47.[Medline]
De Maria, N., Colantoni, A., Fagiuoli, S., Liu, G.-J., Rogers, B. K., Farinati, F., VanThiel, D. H. & Floyd, R. A. (1996). Association between reactive oxygen species and disease activity in chronic hepatitis C. Free Radic Biol Med 21, 291–295.[CrossRef][Medline]
Dhillon, A. P. & Dusheiko, G. M. (1995). Pathology of hepatitis C virus infection. Histopathology 26, 297–309.[Medline]
Dumas, E., Staedel, C., Colombat, M., Reigadas, S., Chabas, S., Astier-Gin, T., Cahour, A., Litvak, S. & Ventura, M. (2003). A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5' UTR. Nucleic Acids Res 31, 1275–1281.
Fernandez, J., Bode, B., Koromilas, A., Diehl, J. A., Krukovets, I., Snider, M. D. & Hatzoglou, M. (2002a). Translation mediated by the internal ribosome entry site of the cat-1 mRNA is regulated by glucose availability in a PERK kinase-dependent manner. J Biol Chem 277, 11780–11787.
Fernandez, J., Yaman, I., Sarnow, P., Snider, M. D. & Hatzoglou, M. (2002b). Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2. J Biol Chem 277, 19198–19205.
Gerlitz, G., Jagus, R. & Elroy-Stein, O. (2002). Phosphorylation of initiation factor-2 is required for activation of internal translation initiation during cell differentiation. Eur J Biochem 269, 2810–2819.[Medline]
Gong, G., Waris, G., Tanveer, R. & Siddiqui, A. (2001). Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-
B. Proc Natl Acad Sci U S A 98, 9599–9604.
Han, B. & Zhang, J.-T. (2002). Regulation of gene expression by internal ribosome entry sites or cryptic promoters: the eIF4G story. Mol Cell Biol 22, 7372–7384.
He, Y., Yan, W., Coito, C., Li, Y., Gale, M., Jr & Katze, M. G. (2003). The regulation of hepatitis C virus (HCV) internal ribosome-entry site-mediated translation by HCV replicons and nonstructural proteins. J Gen Virol 84, 535–543.
Hellen, C. U. T. & Sarnow, P. (2001). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev 15, 1593–1612.
Holcik, M., Sonenberg, N. & Korneluk, R. G. (2000). Internal ribosome initiation of translation and the control of cell death. Trends Genet 16, 469–473.[CrossRef][Medline]
Ito, T., Tahara, S. M. & Lai, M. M. C. (1998). The 3'-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site. J Virol 72, 8789–8796.
Kalliampakou, K. I., Kalamvoki, M. & Mavromara, P. (2005). Hepatitis C virus (HCV) NS5A protein downregulates HCV IRES-dependent translation. J Gen Virol 86, 1015–1025.
Kato, N., Hijikata, M., Ootsuyama, Y., Nakagawa, M., Ohkoshi, S., Sugimura, T. & Shimotonho, K. (1990). Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A 87, 9524–9528.
Korenaga, M., Wang, T., Li, Y., Showalter, L. A., Chan, T., Sun, J. & Weinman, S. A. (2005). Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production. J Biol Chem 280, 37481–37488.
Koumenis, C., Naczki, C., Koritzinsky, M., Rastani, S., Diehl, A., Sonenberg, N., Koromilas, A. & Wouters, B. G. (2002). Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2. Mol Cell Biol 22, 7405–7416.
Krüger, M., Beger, C., Li, Q.-X., Welch, P. J., Tritz, R., Leavitt, M., Barber, J. R. & Wong-Staal, F. (2000). Identification of eIF2B and eIF2 as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach. Proc Natl Acad Sci U S A 97, 8566–8571.
Lauer, G. M. & Walker, B. D. (2001). Hepatitis C virus infection. N Engl J Med 345, 41–52.
Li, D., Takyar, S. T., Lott, W. B. & Gowans, E. J. (2003). Amino acids 1–20 of the hepatitis C virus (HCV) core protein specifically inhibit HCV IRES-dependent translation in HepG2 cells, and inhibit both HCV IRES-mediated and cap-dependent translation in HuH7 and CV-1 cells. J Gen Virol 84, 815–825.
Lohmann, V., Körner, F., Koch, J.-O., Herian, U., Theilmann, L. & Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113.
Moradpour, D., Brass, V., Gosert, R., Wölk, B. & Blum, H. E. (2002). Hepatitis C: molecular virology and antiviral targets. Trends Mol Med 8, 476–482.[CrossRef][Medline]
Moriya, K., Nakagawa, K., Santa, T. & 11 other authors (2001). Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis. Cancer Res 61, 4365–4370.
Novoa, I., Zeng, H., Harding, H. P. & Ron, D. (2001). Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2. J Cell Biol 153, 1011–1022.
Okuda, M., Li, K., Beard, M. R., Showalter, L. A., Scholle, F., Lemon, S. M. & Weinman, S. A. (2002). Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology 122, 366–375.[CrossRef][Medline]
O'Loghlen, A., Pérez-Morgado, M. I., Salinas, M. & Martín, M. E. (2003). Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide. Potential regulation of eIF2 phosphorylation in differentiated PC12 cells. Arch Biochem Biophys 417, 194–202.[CrossRef][Medline]
Paradis, V., Mathurin, P., Kollinger, M. & 7 other authors (1997). In situ detection of lipid peroxidation in chronic hepatitis C: correlation with pathological features. J Clin Pathol 50, 401–406.
Pestova, T. V., Shatsky, I. N., Fletcher, S. P., Jackson, R. T. & Hellen, C. U. T. (1998). A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev 12, 67–83.
Qadri, I., Iwahashi, M., Capasso, J. M., Hopken, M. W., Flores, S., Schaack, J. & Simon, F. R. (2004). Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1. Biochem J 378, 919–928.[CrossRef][Medline]
Reynolds, J. E., Kaminski, A., Kettinen, H. J., Grace, K., Clarke, B. E., Carroll, A. R., Rowlands, D. J. & Jackson, R. J. (1995). Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J 14, 6010–6020.[Medline]
Rivas-Estilla, A. M., Svitkin, Y., Lastra, M. L., Hatzoglou, M., Sherker, A. & Koromilas, A. E. (2002). PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone. J Virol 76, 10637–10653.
Schwarz, K. B. (1996). Oxidative stress during viral infection: a review. Free Radic Biol Med 21, 641–649.[CrossRef][Medline]
Sherrill, K. W., Byrd, M. P., Van Eden, M. E. & Lloyd, R. E. (2004). BCL-2 translation is mediated via internal ribosome entry during cell stress. J Biol Chem 279, 29066–29074.
Tsukiyama-Kohara, K., Iizuka, N., Kohara, M. & Nomoto, A. (1992). Internal ribosome entry site within hepatitis C virus RNA. J Virol 66, 1476–1483.
Van Eden, M. E., Byrd, M. P., Sherrill, K. W. & Lloyd, R. E. (2004a). Demonstrating internal ribosome entry sites in eukaryotic mRNAs using stringent RNA test procedures. RNA 10, 720–730.
Van Eden, M. E., Byrd, M. P., Sherrill, K. W. & Lloyd, R. E. (2004b). Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA 10, 469–481.
Wakita, T., Pietschmann, T., Kato, T. & 9 other authors (2005). Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11, 791–796.[CrossRef][Medline]
Wang, H. & Joseph, J. A. (1999). Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27, 612–616.[CrossRef][Medline]
Yamashita, T., Kaneko, S., Hashimato, S., Sato, T., Nagai, S., Toyoda, N., Suzuki, T., Kobayashi, K. & Matsushima, K. (2001). Serial analysis of gene expression in chronic hepatitis C and hepatocellular carcinoma. Biochem Biophys Res Commun 282, 647–654.[CrossRef][Medline]
Received 20 March 2006;
accepted 10 July 2006.
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