Jinah Choi

Synthesis of a novel hepatitis C virus protein by ribosomal frameshift

Hepatitis C virus (HCV) is an important human pathogen that affects ∼100 million people worldwide. Its RNA genome codes for a polyprotein, which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products. We report here the discovery of a novel HCV protein synthesized by ribosomal frameshift. This protein, which we named the F protein, is synthesized from the initiation codon of the polyprotein sequence followed by ribosomal frameshift into the −2/+1 reading frame. This ribosomal frameshift requires only codons 8–14 of the core protein‐coding sequence, and the shift junction is located at or near codon 11. An F protein analog synthesized in vitro reacted with the sera of HCV patients but not with the sera of hepatitis B patients, indicating the expression of the F protein during natural HCV infection. This unexpected finding may open new avenues for the development of anti‐HCV drugs.

Induction of Incomplete Autophagic Response by Hepatitis C Virus via the Unfolded Protein Response

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. (HEPATOLOGY 2008.)

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‐α), interleukin 6, interleukin 8, tumor growth factor beta, and Fas ligand. TNF‐α 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. (HEPATOLOGY 2008.)

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. (HEPATOLOGY 2004;39:81–89.)

γ‐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.

Age-associated decline in γ-glutamylcysteine synthetase gene expression in rats

Abstract Although glutathione (GSH) concentration has been reported to diminish with age, the mechanism underlying such age-associated decline in the GSH content is not well understood. In this study, we compared the gene expression of both subunits of γ-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, in young, adult, and old Fisher 344 rats. It was found that GCS activity was significantly decreased with increased age in liver, kidney, lung, and red blood cells (RBC). Parallel with the decreased enzyme activity, the protein and mRNA contents of both GCS subunits also changed inversely with age in liver, kidney, and lung, implying a decreased GCS gene expression during aging. Such a reduced GCS gene expression was accompanied by a decline in total GSH content without any change in cysteine concentration. Furthermore, the decreased GCS gene expression in old rats was not associated with a decline in the plasma insulin or cortisol level. This study showed, for the first time, that the expression of both GCS subunit genes was decreased in some organs of old rats, which would result in a reduced rate of GSH biosynthesis. Such decline in GSH synthetic capacity may underlie the observed decrease in GSH content during aging.

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

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 (TGFβ1). TGFβ1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGFβ1. 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. Hepatology 2010

γ-Glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by 4-hydroxy-2-nonenal

γ-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo glutathione (GSH) synthesis, is composed of one catalytic (heavy) and one regulatory (light) subunit. Although both subunits are increased at the mRNA level by oxidants, it is not clear whether they are regulated through the same mechanism. 4-Hydroxy-2-nonenal (4HNE), a lipid peroxidation product, may act as a mediator for the induction of gene expression by oxidants. In the present study, 4HNE was used to study the mechanism of induction of the two GCS subunits in rat lung epithelial L2 cells. 4HNE increased both the transcription rates and the stability of mRNA for both GCS subunits, resulting in an increased mRNA content for both subunits. Both GCS subunit proteins and enzymatic activities also increased. Emetine, a protein synthesis inhibitor, blocked the increase in GCS light subunit mRNA but not the increase in GCS heavy subunit mRNA. This suggested that although 4HNE increased transcription and stabilization of both GCS subunit mRNAs, the signaling pathways involved in the induction of the two GCS subunits differed.

Oxidative Modification of Nuclear Mitogen-activated Protein Kinase Phosphatase 1 Is Involved in Transforming Growth Factor β1-induced Expression of Plasminogen Activator Inhibitor 1 in Fibroblasts

Transforming growth factor β (TGF-β) stimulates reactive oxygen species (ROS) production in various cell types, which mediates many of the effects of TGF-β. The molecular mechanisms whereby TGF-β increases ROS production and ROS modulate the signaling processes of TGF-β, however, remain poorly defined. In this study, we show that TGF-β1 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-β1-induced phosphorylation of JNK and p38 as well as the expression of plasminogen activator inhibitor 1 (PAI-1), a TGF-β-responsive gene involved in the pathogenesis of many diseases. Knockdown of Nox4 with Nox4 small interfering RNA, on the other hand, reduces TGF-β1-stimulated ROS production, p38 phosphorylation, and PAI-1 expression. TGF-β also increased the nuclear level of Nox4 protein as well as PAI-1 expression in human lung fibroblasts (CCL-210 cells), suggesting that TGF-β 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-β signaling pathways. Our data suggest that increased generation of ROS by Nox4 mediates TGF-β1-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.

Hepatitis C Virus F Protein Is a Short-Lived Protein Associated with the Endoplasmic Reticulum

ABSTRACT Hepatitis C virus (HCV) F protein is a newly discovered HCV gene product that is expressed by translational ribosomal frameshift. Little is known about the biological properties of this protein. By performing pulse-chase labeling experiments, we demonstrate here that the F protein is a labile protein with a half-life of <10 min in Huh7 hepatoma cells and in vitro. The half-life of the F protein could be substantially increased by proteasome inhibitors, suggesting that the rapid degradation of the F protein is mediated by the proteasome pathway. Further immunofluorescence staining and subcellular fractionation experiments indicate that the F protein is primarily associated with the endoplasmic reticulum. This subcellular localization is similar to those of HCV core and NS5A proteins, raising the possibility that the F protein may participate in HCV morphogenesis or replication.