Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancer-related mortality in the world. Hepatitis C virus (HCV) is a major etiologic agent of HCC. A majority of HCV infections lead to chronic infection that can progress to cirrhosis and, eventually, HCC and liver failure. A common pathogenic feature present in HCV infection, and other conditions leading to HCC, is oxidative stress. HCV directly increases superoxide and H2O2 formation in hepatocytes by elevating Nox protein expression and sensitizing mitochondria to reactive oxygen species generation while decreasing glutathione. Nitric oxide synthesis and hepatic iron are also elevated. Furthermore, activation of phagocytic NADPH oxidase (Nox) 2 of host immune cells is likely to exacerbate oxidative stress in HCV-infected patients. Key mechanisms of HCC include genome instability, epigenetic regulation, inflammation with chronic tissue injury and sustained cell proliferation, and modulation of cell growth and death. Oxidative stress, or Nox proteins, plays various roles in these mechanisms. Nox proteins also function in hepatic fibrosis, which commonly precedes HCC, and Nox4 elevation by HCV is mediated by transforming growth factor ß. This review summarizes mechanisms of oncogenesis by HCV, highlighting the roles of oxidative stress and hepatic Nox enzymes in HCC.

Oxidative stress, endogenous antioxidants, alcohol, and hepatitis C: pathogenic interactions and therapeutic considerations.

Hepatitis C virus (HCV) is a blood-borne pathogen that was identified as an etiologic agent of non-A, non-B hepatitis in 1989. HCV is estimated to have infected at least 170 million people worldwide. The majority of patients infected with HCV do not clear the virus and become chronically infected, and chronic HCV infection increases the risk for hepatic steatosis, cirrhosis, and hepatocellular carcinoma. HCV induces oxidative/nitrosative stress from multiple sources, including inducible nitric oxide synthase, the mitochondrial electron transport chain, hepatocyte NAD(P)H oxidases, and inflammation, while decreasing glutathione. The cumulative oxidative burden is likely to promote both hepatic and extrahepatic conditions precipitated by HCV through a combination of local and more distal effects of reactive species, and clinical, animal, and in vitro studies strongly point to a role of oxidative/nitrosative stress in HCV-induced pathogenesis. Oxidative stress and hepatopathogenesis induced by HCV are exacerbated by even low doses of alcohol. Alcohol and reactive species may have other effects on hepatitis C patients such as modulation of the host immune system, viral replication, and positive selection of HCV sequence variants that contribute to antiviral resistance. This review summarizes the current understanding of redox interactions of HCV, outlining key experimental findings, directions for future research, and potential applications to therapy.

Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals.

Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4-18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance.

Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection.

UNLABELLED: Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV)-induced pathogenesis. Studies have suggested that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increases the generation of superoxide and hydrogen peroxide close to the hepatocyte nucleus and that this source of ROS is reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 4 (Nox4). Huh7 human hepatoma cells and telomerase-reconstituted primary human hepatocytes, transfected or infected with virus-producing HCV strains of genotypes 2a and 1b, were examined for messenger RNA (mRNA), protein, and subcellular localization of Nox proteins along with the human liver. We found that genotype 2a HCV induced persistent elevations of Nox1 and Nox4 mRNA and proteins in Huh7 cells. HCV genotype 1b likewise elevated the levels of Nox1 and Nox4 in telomerase-reconstituted primary human hepatocytes. Furthermore, Nox1 and Nox4 proteins were increased in HCV-infected human liver versus uninfected liver samples. Unlike Nox1, Nox4 was prominent in the nuclear compartment of these cells as well as the human liver, particularly in the presence of HCV. HCV-induced ROS and nuclear nitrotyrosine could be decreased with small interfering RNAs to Nox1 and Nox4. Finally, HCV increased the level of transforming growth factor beta 1 (TGFbeta1). TGFbeta1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGFbeta1. CONCLUSION: HCV induced a persistent elevation of Nox1 and Nox4 and increased nuclear localization of Nox4 in hepatocytes in vitro and in the human liver. Hepatocyte Nox proteins are likely to act as a persistent, endogenous source of ROS during HCV-induced pathogenesis.

Oxidative modification of nuclear mitogen-activated protein kinase phosphatase 1 is involved in transforming growth factor beta1-induced expression of plasminogen activator inhibitor 1 in fibroblasts.

Transforming growth factor beta (TGF-beta) stimulates reactive oxygen species (ROS) production in various cell types, which mediates many of the effects of TGF-beta. The molecular mechanisms whereby TGF-beta increases ROS production and ROS modulate the signaling processes of TGF-beta, however, remain poorly defined. In this study, we show that TGF-beta1 stimulates NADPH oxidase 4 (Nox4) expression and ROS generation in the nucleus of murine embryo fibroblasts (NIH3T3 cells). This is associated with an increase in protein thiol modification and inactivation of MAPK phosphatase 1 (MKP-1), a nuclear phosphatase. Furthermore, knockdown of MKP-1 using small interfering RNA enhances TGF-beta1-induced phosphorylation of JNK and p38 as well as the expression of plasminogen activator inhibitor 1 (PAI-1), a TGF-beta-responsive gene involved in the pathogenesis of many diseases. Knockdown of Nox4 with Nox4 small interfering RNA, on the other hand, reduces TGF-beta1-stimulated ROS production, p38 phosphorylation, and PAI-1 expression. TGF-beta also increased the nuclear level of Nox4 protein as well as PAI-1 expression in human lung fibroblasts (CCL-210 cells), suggesting that TGF-beta may induce PAI-1 expression by a similar mechanism in human lung fibroblasts. In summary, in this study we have identified nuclear MAPK phosphatase MKP-1 as a novel molecular target of ROS in TGF-beta signaling pathways. Our data suggest that increased generation of ROS by Nox4 mediates TGF-beta1-induced PAI-1 gene expression at least in part through oxidative modification and inhibition of MKP-1 leading to a sustained activation of JNK and p38 MAPKs.

Ethanol enhances hepatitis C virus replication through lipid metabolism and elevated NADH/NAD+.

UNLABELLED: Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD(+) ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD(+). Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting beta-oxidation with beta-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. CONCLUSION: Therefore, lipid metabolism and alteration of cellular NADH/NAD(+) ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress.

Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response.

UNLABELLED: Autophagy is important for cellular homeostasis and can serve as innate immunity to remove intracellular pathogens. Here, we demonstrate by a battery of morphological and biochemical assays that hepatitis C virus (HCV) induces the accumulation of autophagosomes in cells without enhancing autophagic protein degradation. This induction of autophagosomes depended on the unfolded protein response (UPR), as the suppression of UPR signaling pathways suppressed HCV-induced lipidation of the microtubule-associated protein light chain 3 (LC3) protein, a necessary step for the formation of autophagosomes. The suppression of UPR or the suppression of expression of LC3 or Atg7, a protein that mediates LC3 lipidation, suppressed HCV replication, indicating a positive role of UPR and the incomplete autophagic response in HCV replication. CONCLUSION: Our studies delineate the molecular pathway by which HCV induces autophagic vacuoles and also demonstrate the perturbation of the autophagic response by HCV. These unexpected effects of HCV on the host cell likely play an important role in HCV pathogenesis.

Hyperthermic stress-induced increase in the expression of glutamate-cysteine ligase and glutathione levels in the symbiotic sea anemone Aiptasia pallida.

Hyperthermic stress is known to trigger the loss of unicellular algae from a number of symbiotic cnidarians, a phenomenon commonly referred to as bleaching. Oxidative and nitrosative stress have been suggested to play a major role during the process of bleaching, however the underlying molecular mechanisms are still poorly understood. In animals, the intracellular tripeptide glutathione (GSH) is involved in antioxidant defense, redox homeostasis and intracellular redox signaling. Therefore, we tested the hypothesis that hyperthermal stress-induced bleaching in Aiptasia pallida, a model for symbiotic cnidarians, results in increased levels of GSH synthesis. We report the cDNA sequence and functional analysis of the catalytic subunit of glutamate-cysteine ligase (GCLC), which catalyzes the rate-limiting step in GSH biosynthesis. In a time-series experiment, both GCLC gene expression and total GSH levels increased 4- and 1.5-fold, respectively, in response to hyperthermal stress. These results suggest that hyperthermal stress triggers adaptive increases in intracellular GSH biosynthesis in cnidarians as a protective response to oxidative/nitrosative stress. Our results show the conserved function of GCLC and GSH across animals while placing a new perspective on the role of GSH in redox signaling during cnidarian bleaching.

Hepatitis C virus infection: molecular pathways to metabolic syndrome.

Chronic infection with hepatitis C virus (HCV) can induce insulin resistance (IR) in a genotype-dependent fashion, thus contributing to steatosis, progression of fibrosis and resistance to interferon therapy. The molecular mechanisms in genotype 1 patients that lead to metabolic syndrome are still ambiguous. Based on our current understanding, HCV proteins associate with mitochondria and endoplasmic reticulum and promote oxidative stress. The latter mediates signals involving the p38 mitogen-activated protein kinase and activates nuclear factor kappa B. This transcription factor plays a key role in the expression of cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin 6, interleukin 8, tumor growth factor beta, and Fas ligand. TNF-alpha inhibits the function of insulin receptor substrates and decreases the expression of the glucose transporter and lipoprotein lipase in peripheral tissues, which is responsible for the promotion of insulin resistance. Furthermore, reduced adiponectin levels, loss of adiponectin receptors, and decreased anti-inflammatory peroxisome proliferator-activated receptor alpha in the liver of HCV patients may contribute to reduced fatty acid oxidation, inflammation, and eventually lipotoxicity. This chain of events may be initiated by HCV-associated IR and provides a direction for future research in the areas of therapeutic intervention.

Inducible alterations of glutathione levels in adult dopaminergic midbrain neurons result in nigrostriatal degeneration.

Parkinson's disease is a neurodegenerative disorder characterized by the preferential loss of midbrain dopaminergic neurons in the substantia nigra (SN). One of the earliest detectable biochemical alterations that occurs in the Parkinsonian brain is a marked reduction in SN levels of total glutathione (glutathione plus glutathione disulfide), occurring before losses in mitochondrial complex I (CI) activity, striatal dopamine levels, or midbrain dopaminergic neurodegeneration associated with the disease. Previous in vitro data from our laboratory has suggested that prolonged depletion of dopaminergic glutathione results in selective impairment of mitochondrial complex I activity through a reversible thiol oxidation event. To address the effects of depletion in dopaminergic glutathione levels in vivo on the nigrostriatal system, we created genetically engineered transgenic mouse lines in which expression of gamma-glutamyl cysteine ligase, the rate-limiting enzyme in de novo glutathione synthesis, can be inducibly downregulated in catecholaminergic neurons, including those of the SN. A novel method for isolation of purified dopaminergic striatal synaptosomes was used to study the impact of dopaminergic glutathione depletion on mitochondrial events demonstrated previously to occur in vitro as a consequence of this alteration. Dopaminergic glutathione depletion was found to result in a selective reversible thiol-oxidation-dependent mitochondrial complex I inhibition, followed by an age-related nigrostriatal neurodegeneration. This suggests that depletion in glutathione within dopaminergic SN neurons has a direct impact on mitochondrial complex I activity via increased nitric oxide-related thiol oxidation and age-related dopaminergic SN cell loss.

Glutathione suppresses TGF-beta-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter.

Transforming growth factor (TGF)-beta upregulates plasminogen activator inhibitor type 1 (PAI-1) in a variety of cell types, and PAI-1 is considered to be an essential factor for the development of fibrosis. Our previous studies demonstrated that TGF-beta decreased intracellular glutathione (GSH) content in murine embryonic fibroblasts (NIH/3T3 cells), whereas treatment of the cells with GSH, which restored intracellular GSH concentration, inhibited TGF-beta-induced collagen accumulation by blocking PAI-1 expression and enhancing collagen degradation. In the present study, we demonstrate that GSH blocks TGF-beta-induced PAI-1 promoter activity in NIH/3T3 cells, which is associated with an inhibition of TGF-beta-induced JNK and p38 phosphorylation. Interestingly, although exogenous GSH does not affect phosphorylation and/or nuclear translocation of Smad2/3 and Smad4, it completely eliminates TGF-beta-induced binding of transcription factors to not only AP-1 and SP-1 but also Smad cis elements in the PAI-1 promoter. Decoy oligonucleotides (ODN) studies further demonstrate that AP-1, SP-1, and Smad ODNs abrogate the inhibitory effect of GSH on TGF-beta-induced PAI-1 promoter activity and inhibit TGF-beta-induced expression of endogenous PAI-1. Furthermore, we show that GSH reduces TGF-beta-stimulated reactive oxygen species (ROS) signal. Blocking ROS production with diphenyleneiodonium or scavenging ROS with a superoxide dismutase and catalase mimetic MnTBaP dramatically reduces TGF-beta-induced p38 and JNK phosphorylation as well as PAI-1 gene expression. In composite, these findings suggest that GSH inhibits TGF-beta-stimulated PAI-1 expression in fibroblasts by blocking the JNK/p38 pathway, probably by reducing ROS, which leads to an inhibition of the binding of transcription factors to the AP-1, SP-1, and Smad cis elements in the PAI-1 promoter.

Redox regulation of hepatitis C in nonalcoholic and alcoholic liver.

Hepatitis C virus (HCV) is an RNA virus of the Flaviviridae family that is estimated to have infected 170 million people worldwide. HCV can cause serious liver disease in humans, such as cirrhosis, steatosis, and hepatocellular carcinoma. HCV induces a state of oxidative/nitrosative stress in patients through multiple mechanisms, and this redox perturbation has been recognized as a key player in HCV-induced pathogenesis. Studies have shown that alcohol synergizes with HCV in the pathogenesis of liver disease, and part of these effects may be mediated by reactive species that are generated during hepatic metabolism of alcohol. Furthermore, reactive species and alcohol may influence HCV replication and the outcome of interferon therapy. Alcohol consumption has also been associated with increased sequence heterogeneity of the HCV RNA sequences, suggesting multiple modes of interaction between alcohol and HCV. This review summarizes the current understanding of oxidative and nitrosative stress during HCV infection and possible combined effects of HCV, alcohol, and reactive species in the pathogenesis of liver disease.

Redox modulation of the hepatitis C virus replication complex is calcium dependent.

Reactive species and perturbation of the redox balance have been implicated in the pathogenesis of many viral diseases, including hepatitis C. Previously, we made a surprising discovery that concentrations of H(2)O(2) that are nontoxic to host cells disrupted the hepatitis C virus (HCV) replication complex (RC) in Huh7 human hepatoma cells in a manner that suggested signaling. Here, we show that H(2)O(2) and interferon-gamma have comparable effects on the HCV subgenomic and genomic RNA replication in Huh7 cells. H(2)O(2) induced a gradual rise in the intracellular calcium concentration ([Ca(2+)](i)). Both rapid and sustained suppression of HCV RNA replication by H(2)O(2) depended on this calcium elevation. The peroxide-induced [Ca(2+)](i) elevation was independent of extracellular calcium and derived, at least in part, from the endoplasmic reticulum. Likewise, the suppression of the HCV RC by H(2)O(2) was independent of extracellular calcium but required an intracellular calcium source. Other agents that elevated [Ca(2+)](i) could also suppress the HCV RC, suggesting that calcium elevation might be sufficient to suppress HCV RNA replication. In conclusion, oxidants may modulate the HCV RC through calcium. Effects on the infectivity and the morphogenesis of HCV remain to be determined. These findings suggest possible regulatory roles for redox and calcium signaling during viral infections.

Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus.

Hepatitis C virus (HCV) is a major cause of viral hepatitis that can progress to hepatic fibrosis, steatosis, hepatocellular carcinoma, and liver failure. HCV infection is characterized by a systemic oxidative stress that is most likely caused by a combination of chronic inflammation, iron overload, liver damage, and proteins encoded by HCV. The increased generation of reactive oxygen and nitrogen species, together with the decreased antioxidant defense, promotes the development and progression of hepatic and extrahepatic complications of HCV infection. This review discusses the possible mechanisms of HCV-induced oxidative stress and its role in HCV pathogenesis.

Nitric oxide-induced resistance to hydrogen peroxide stress is a glutamate cysteine ligase activity-dependent process.

Nitric oxide (*NO) is a reactive nitrogen species known to be involved in cytotoxic processes. Cells respond to cytotoxic injury by stress response induction leading to the development of cellular resistance. This report describes an *NO-induced stress response in Chinese hamster fibroblasts (HA1), which leads to glutathione synthesis-dependent resistance to H2O2-mediated oxidative stress. The development of resistance to H2O2 was completely abolished by the inhibition of glutamate cysteine ligase (GCL) during the first 8 h of recovery after *NO exposure. Altered thiol metabolism was observed immediately after *NO exposure as demonstrated by up to 75% decrease in intracellular thiol pools (glutathione, gamma-glutamylcysteine, and cysteine), which then reaccumulated during the *NO-mediated development of resistance. Immunoreactive protein and activity associated with GCL decreased immediately after exposure to *NO and then reaccumulated during the development of resistance to H2O2 challenge. Moreover, compared to N2 controls the activity levels of GCL in *NO-exposed cells increased approximately twofold 24 h after H2O2 challenge. These results demonstrate that *NO exposure is capable of inducing an adaptive response to H2O2-mediated oxidative stress in mammalian cells, which involves alterations in thiol metabolism and is dependent upon glutathione synthesis and increased GCL activity.

Gamma-glutamyl transpeptidase in glutathione biosynthesis.

Glutathione (GSH) is the most abundant nonprotein thiol in cells and has multiple biological functions. Glutathione biosynthesis by way of the gamma-glutamyl cycle is important for maintaining GSH homeostasis and normal redox status. As the only enzyme of the cycle located on the outer surface of plasma membrane, gamma-glutamyl transpeptidase (GGT) plays key roles in GSH homeostasis by breaking down extracellular GSH and providing cysteine, the rate-limiting substrate, for intracellular de novo synthesis of GSH. GGT also initiates the metabolism of glutathione S-conjugates to mercapturic acids by transferring the gamma-glutamyl moiety to an acceptor amino acid and releasing cysteinylglycine. GGT is expressed in a tissue-, developmental phase-, and cell-specific manner that may be related to its complex gene structure. In rodents, there is a single GGT gene, and several promoters that generate different mRNA subtypes and regulate its expression. In contrast, several GGT genes have been found in humans. During oxidative stress, GGT gene expression is increased, and this is believed to constitute an adaptation to stress. Interestingly, only certain mRNA subtypes are increased, suggesting a specific mode of regulation of GGT gene expression by oxidants. Here, protocols to measure GGT activity, relative levels of total and specific GGT mRNA subtypes, and GSH concentration are described.

The C-terminal transmembrane domain of hepatitis C virus (HCV) RNA polymerase is essential for HCV replication in vivo.

Hepatitis C virus (HCV) RNA replication is dependent on the enzymatic activities of the viral RNA-dependent RNA polymerase NS5B, which is a membrane-anchored protein. Recombinant NS5B lacking the C-terminal transmembrane domain (21 amino acids) is enzymatically active. To address the role of this domain in HCV replication in vivo, we introduced a series of mutations into the NS5B of an HCV subgenomic replicon and examined the replication capabilities of the resultant mutants by a colony formation assay. Replicons lacking the transmembrane domain did not yield any colonies. Furthermore, when Huh-7 cells harboring the HCV subgenomic replicon were treated with a synthetic peptide consisting of the NS5B transmembrane domain fused to the antennapedia peptide, the membrane association of NS5B was completely disrupted. Correspondingly, the HCV RNA titer was reduced by approximately 50%. A scrambled peptide used as a control did not have any effects. These findings suggest that the membrane association of NS5B facilitates HCV RNA synthesis. However, a related transmembrane domain derived from bovine viral diarrhea virus could not replace the HCV NS5B transmembrane segment. This finding suggests that the C-terminal 21 amino acids not only have a membrane-anchoring function but also may perform additional functions for RNA synthesis in vivo.

Reactive oxygen species suppress hepatitis C virus RNA replication in human hepatoma cells.

Hepatitis C virus (HCV) is a positive-stranded RNA virus that causes severe liver diseases, such as cirrhosis and hepatocellular carcinoma. HCV uses an RNA-dependent RNA polymerase to replicate its genome and an internal ribosomal entry site to translate its proteins. HCV infection is characterized by an increase in the concentrations of reactive oxygen species (ROS), the effect of which on HCV replication has yet to be determined. In this report, we investigated the effect of ROS on HCV replication, using a bicistronic subgenomic RNA replicon and a genomic RNA that can replicate in human hepatoma cells. The treatment with peroxide at concentrations that did not deplete intracellular glutathione or induce cell death resulted in significant decreases in the HCV RNA level in the cells. This response could be partially reversed by the antioxidant N-acetylcysteine. Further studies indicated that such a suppressive response to ROS was not due to the suppression of HCV protein synthesis or the destabilization of HCV RNA. Rather, it occurred rapidly at the level of RNA replication. ROS appeared to disrupt active HCV replication complexes, as they reduced the amount of NS3 and NS5A in the subcellular fraction where active HCV RNA replication complexes were found. In conclusion, our results show that ROS can rapidly inhibit HCV RNA replication in human hepatoma cells. The increased ROS levels in hepatitis C patients may therefore play an important role in the suppression of HCV replication.

Triple decoding of hepatitis C virus RNA by programmed translational frameshifting.

Ribosomes can be programmed to shift from one reading frame to another during translation. Hepatitis C virus (HCV) uses such a mechanism to produce F protein from the -2/+1 reading frame. We now report that the HCV frameshift signal can mediate the synthesis of the core protein of the zero frame, the F protein of the -2/+1 frame, and a 1.5-kDa protein of the -1/+2 frame. This triple decoding function does not require sequences flanking the frameshift signal and is apparently independent of membranes and the synthesis of the HCV polyprotein. Two consensus -1 frameshift sequences in the HCV type 1 frameshift signal facilitate ribosomal frameshifts into both overlapping reading frames. A sequence which is located immediately downstream of the frameshift signal and has the potential to form a double stem-loop structure can significantly enhance translational frameshifting in the presence of the peptidyl-transferase inhibitor puromycin. Based on these results, a model is proposed to explain the triple decoding activities of the HCV ribosomal frameshift signal.