Hepatitis C Virus (HCV) Constitutively Activates S
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病菌学杂志 2005年第3期
Department of Microbiology and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado
Molecular Oncology Program, Moffitt Cancer Center, Tampa, Florida
Department of Clinical Medicine and Surgery, School of Medicine, University of California, San Diego, California
ABSTRACT
The hepatitis C virus (HCV) causes chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. We have previously shown that HCV nonstructural proteins induce activation of STAT-3 via oxidative stress and Ca2+ signaling (G. Gong, G. Waris, R. Tanveer, and A. Siddiqui, Proc. Natl. Acad. Sci. USA 98:9599-9604, 2001). In this study, we focus on the signaling pathway leading to STAT-3 activation in response to oxidative stress induced by HCV translation and replication activities. Here, we demonstrate the constitutive activation of STAT-3 in HCV replicon-expressing cells. The HCV-induced STAT-3 activation was inhibited in the presence of antioxidant (pyrrolidine dithiocarbamate) and Ca2+ chelators (BAPTA-AM and TMB-8). Previous studies have shown that maximum STAT-3 transactivation requires Ser727 phosphorylation in addition to tyrosine phosphorylation. Using a series of inhibitors and dominant negative mutants, we show that HCV-induced activation of STAT-3 is mediated by oxidative stress and influenced by the activation of cellular kinases, including p38 mitogen-activated protein kinase, JNK, JAK-2, and Src. Our results also suggest a potential role of STAT-3 in HCV RNA replication. We also observed the constitutive activation of STAT-3 in the liver biopsy of an HCV-infected patient. These studies provide an insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with the viral infection.
INTRODUCTION
The hepatitis C virus (HCV) is one of the leading causes of chronic liver disease, afflicting more than 170 million individuals worldwide (17). Persistent HCV infection often leads to a risk of end-stage cirrhosis and hepatocellular carcinoma (17). HCV is an enveloped single-stranded positive-sense RNA virus, approximately 9.6 kb in length, and encodes a polyprotein of about 3,000 amino acids (18). This polyprotein is posttranslationally cleaved by a combination of host cell signal peptidases and two viral proteinases into structural (core, E1, and E2) and nonstructural (NS2 and NS3-NS5A/B) proteins (2, 36). A new protein termed F is thought to be produced by –2/+1 ribosomal frameshift during translation (55). The single open reading frame is flanked at the 5' end by a noncoding region (NCR), which harbors an internal ribosome entry site (46, 49) and at the 3' end by a highly conserved sequence essential for initiating RNA replication (44).
Despite the availability of infectious cDNA clones (27), molecular studies of HCV replication and pathogenesis have been hampered by the lack of a reliable and efficient cell culture system. To overcome these restrictions recently, Lohmann et al. reported the development of HCV subgenomic replicons (29). These bicistronic replicons are composed of an HCV 5' NCR fused to 12 amino acids of the capsid coding region, the neomycin phosphotransferase gene (Neor), which confers resistance to G418, and the internal ribosome entry site from encephalomyocarditis virus, controlling the translation of the HCV proteins NS3 to NS5B, followed by the 3' NCR. Several adaptive mutations were identified scattered throughout the NS proteins of the replicon, which conferred a high level of replication of subgenomic replicons (30). Viral proteins are found exclusively in the cytoplasm in close association with endoplasmic reticulum (ER) membrane, suggesting this as the site of RNA replication (3). A recent study has described the association of RNA replication with lipid rafts (39). The HCV nonstructural proteins form a ribonucleoprotein complex which is localized in the ER membrane (2, 18, 53). This association induces ER stress, exhibiting an unfolded protein response (45). Depletion of Ca2+ stores in the ER and its uptake by mitochondria lead to generation of reactive oxygen species (ROS) (24). Several HCV proteins, including core, NS3, and NS5A, have been shown to induce oxidative stress in cultured cells (7, 24, 34). ROS, which act as second messengers, activate cellular kinases, although the mechanism of this activation is unclear. Some of these kinases can activate transcription factors that are in a latent state in the cytoplasm. These include STAT-3, NF-B, NF-AT, and others (10, 24).
STAT-3 is an oncogenic transcription factor that is activated upon tyrosine phosphorylation in response to extracellular signals, such as cytokines and growth factors (6, 62). Binding of cytokines such as interleukin-6 or growth factors to their cognate receptors leads to receptor dimerization and activation of receptor-associated Janus kinases (JAKs), resulting in recruitment of STAT-3 protein (57). Activated STAT-3 then translocates to the nucleus to regulate gene expression. STAT-3 activation by other nonreceptor tyrosine kinases has also been demonstrated. Transformation of mammalian cells by viral Src (v-Src) specifically induces constitutive activation of STAT-3 (47, 58). In addition to STAT-3 activation by tyrosine phosphorylation, Ser727 phosphorylation mediated by mitogen-activated protein kinases (MAPKs) contributes to its maximal transcriptional activity (48, 54). Recently, the functional importance of STAT-3 Ser727 phosphorylation has been demonstrated in mice lacking STAT-3 serine phosphorylation (38). STAT-3 is frequently overactivated in a wide variety of solid tumors and blood malignancies (59).
In the present study, we investigated the mechanism(s) of STAT-3 activation in response to oxidative stress induced by HCV gene expression in hepatoma cell lines expressing the HCV subgenomic replicon. Our results
Molecular Oncology Program, Moffitt Cancer Center, Tampa, Florida
Department of Clinical Medicine and Surgery, School of Medicine, University of California, San Diego, California
ABSTRACT
The hepatitis C virus (HCV) causes chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. We have previously shown that HCV nonstructural proteins induce activation of STAT-3 via oxidative stress and Ca2+ signaling (G. Gong, G. Waris, R. Tanveer, and A. Siddiqui, Proc. Natl. Acad. Sci. USA 98:9599-9604, 2001). In this study, we focus on the signaling pathway leading to STAT-3 activation in response to oxidative stress induced by HCV translation and replication activities. Here, we demonstrate the constitutive activation of STAT-3 in HCV replicon-expressing cells. The HCV-induced STAT-3 activation was inhibited in the presence of antioxidant (pyrrolidine dithiocarbamate) and Ca2+ chelators (BAPTA-AM and TMB-8). Previous studies have shown that maximum STAT-3 transactivation requires Ser727 phosphorylation in addition to tyrosine phosphorylation. Using a series of inhibitors and dominant negative mutants, we show that HCV-induced activation of STAT-3 is mediated by oxidative stress and influenced by the activation of cellular kinases, including p38 mitogen-activated protein kinase, JNK, JAK-2, and Src. Our results also suggest a potential role of STAT-3 in HCV RNA replication. We also observed the constitutive activation of STAT-3 in the liver biopsy of an HCV-infected patient. These studies provide an insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with the viral infection.
INTRODUCTION
The hepatitis C virus (HCV) is one of the leading causes of chronic liver disease, afflicting more than 170 million individuals worldwide (17). Persistent HCV infection often leads to a risk of end-stage cirrhosis and hepatocellular carcinoma (17). HCV is an enveloped single-stranded positive-sense RNA virus, approximately 9.6 kb in length, and encodes a polyprotein of about 3,000 amino acids (18). This polyprotein is posttranslationally cleaved by a combination of host cell signal peptidases and two viral proteinases into structural (core, E1, and E2) and nonstructural (NS2 and NS3-NS5A/B) proteins (2, 36). A new protein termed F is thought to be produced by –2/+1 ribosomal frameshift during translation (55). The single open reading frame is flanked at the 5' end by a noncoding region (NCR), which harbors an internal ribosome entry site (46, 49) and at the 3' end by a highly conserved sequence essential for initiating RNA replication (44).
Despite the availability of infectious cDNA clones (27), molecular studies of HCV replication and pathogenesis have been hampered by the lack of a reliable and efficient cell culture system. To overcome these restrictions recently, Lohmann et al. reported the development of HCV subgenomic replicons (29). These bicistronic replicons are composed of an HCV 5' NCR fused to 12 amino acids of the capsid coding region, the neomycin phosphotransferase gene (Neor), which confers resistance to G418, and the internal ribosome entry site from encephalomyocarditis virus, controlling the translation of the HCV proteins NS3 to NS5B, followed by the 3' NCR. Several adaptive mutations were identified scattered throughout the NS proteins of the replicon, which conferred a high level of replication of subgenomic replicons (30). Viral proteins are found exclusively in the cytoplasm in close association with endoplasmic reticulum (ER) membrane, suggesting this as the site of RNA replication (3). A recent study has described the association of RNA replication with lipid rafts (39). The HCV nonstructural proteins form a ribonucleoprotein complex which is localized in the ER membrane (2, 18, 53). This association induces ER stress, exhibiting an unfolded protein response (45). Depletion of Ca2+ stores in the ER and its uptake by mitochondria lead to generation of reactive oxygen species (ROS) (24). Several HCV proteins, including core, NS3, and NS5A, have been shown to induce oxidative stress in cultured cells (7, 24, 34). ROS, which act as second messengers, activate cellular kinases, although the mechanism of this activation is unclear. Some of these kinases can activate transcription factors that are in a latent state in the cytoplasm. These include STAT-3, NF-B, NF-AT, and others (10, 24).
STAT-3 is an oncogenic transcription factor that is activated upon tyrosine phosphorylation in response to extracellular signals, such as cytokines and growth factors (6, 62). Binding of cytokines such as interleukin-6 or growth factors to their cognate receptors leads to receptor dimerization and activation of receptor-associated Janus kinases (JAKs), resulting in recruitment of STAT-3 protein (57). Activated STAT-3 then translocates to the nucleus to regulate gene expression. STAT-3 activation by other nonreceptor tyrosine kinases has also been demonstrated. Transformation of mammalian cells by viral Src (v-Src) specifically induces constitutive activation of STAT-3 (47, 58). In addition to STAT-3 activation by tyrosine phosphorylation, Ser727 phosphorylation mediated by mitogen-activated protein kinases (MAPKs) contributes to its maximal transcriptional activity (48, 54). Recently, the functional importance of STAT-3 Ser727 phosphorylation has been demonstrated in mice lacking STAT-3 serine phosphorylation (38). STAT-3 is frequently overactivated in a wide variety of solid tumors and blood malignancies (59).
In the present study, we investigated the mechanism(s) of STAT-3 activation in response to oxidative stress induced by HCV gene expression in hepatoma cell lines expressing the HCV subgenomic replicon. Our results