Soon B. Hwang

Hepatitis C Virus RNA Replication Occurs on a Detergent-Resistant Membrane That Cofractionates with Caveolin-2

ABSTRACT The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly understood. In this study, we labeled de novo-synthesized viral RNA in situ with bromouridine triphosphate (BrUTP) in Huh7 cells expressing an HCV subgenomic replicon. By immunofluorescence staining using an anti-BrUTP antibody and confocal microscopy, we showed that the newly synthesized HCV RNA was localized to distinct speckle-like structures, which also contain all of the HCV nonstructural (NS) proteins. These speckles are distinct from lipid droplets and are separated from the endoplasmic reticulum (ER), where some HCV NS proteins also reside. Membrane flotation analysis demonstrated that almost all of the NS5A and part of the NS5B proteins and all of the viral RNA were present in membrane fractions which are resistant to treatment with 1% NP-40 at 4°C. They were cofractionated with caveolin-2, a lipid-raft-associated intracellular membrane protein, in the presence or absence of the detergent. In contrast, the ER-resident proteins were detergent soluble. These properties suggest that the membranes on which HCV RNA replication occurs are lipid rafts recruited from the intracellular membranes. The protein synthesis inhibitors cycloheximide and puromycin did not inhibit viral RNA synthesis, indicating that HCV RNA replication does not require continuous protein synthesis. We suggest that HCV RNA synthesis occurs on a lipid raft membrane structure.

Hepatitis C Virus Nonstructural 4B Protein Modulates Sterol Regulatory Element-binding Protein Signaling via the AKT Pathway

Hepatitis C virus (HCV) infection is often associated with hepatic steatosis and yet the molecular mechanisms of HCV-associated steatosis are poorly understood. Because sterol regulatory element-binding proteins (SREBPs) are the major transcriptional factors in lipogenic gene expression including fatty acid synthase (FAS), we examined the effects of HCV nonstructural proteins on the signaling pathways of SREBP. In this study, we demonstrated that HCV nonstructural 4B (NS4B) protein increased the transcriptional activities of SREBPs. We also showed that HCV NS4B enhanced the protein expression levels of SREBPs and FAS. This was further confirmed in the context of viral RNA replication and HCV infection. The up-regulation of both SREBP and FAS by NS4B protein required phosphatidylinositol 3-kinase activity. We also demonstrated that NS4B protein induced a lipid accumulation in hepatoma cells. In addition, NS4B protein synergistically elevated the transcriptional activity of HCV core-mediated SREBP-1. These results strongly suggest that NS4B may play an important role in HCV-associated liver pathogenesis by modulating the SREBP signaling pathway.

Interaction with a Ubiquitin-Like Protein Enhances the Ubiquitination and Degradation of Hepatitis C Virus RNA-Dependent RNA Polymerase

ABSTRACT To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.

Hepatitis C Virus NS5A Protein Interacts with Phosphatidylinositol 4-Kinase Type IIIα and Regulates Viral Propagation

Hepatitis C Virus (HCV) nonstructural 5A (NS5A) is a pleiotropic protein involved in viral RNA replication and modulation of the cellular physiology in HCV-infected cells. To elucidate the mechanisms of the HCV life cycle, we identified cellular factors interacting with the NS5A protein in HCV-infected cells. Huh7.5 cells were electroporated with HCV Jc1 RNA. Cellular factors associated with HCV NS5A were identified by immunoprecipitation with Dynabead-conjugated NS5A antibody and LC-MS/MS. Phosphatidylinositol 4-kinase type IIIα (PI4KIIIα) was identified as a binding partner for the NS5A protein. NS5A derived from both genotypes 1b and 2a interacted with PI4KIIIα. NS5A interacted with PI4KIIIα through amino acids 401–600 of PI4KIIIα and domain I of NS5A. Interference of the protein interaction between NS5A and PI4KIIIα decreased HCV propagation. Knockdown of PI4KIIIα significantly reduced HCV replication in Huh7 cells harboring the subgenomic replicon and in Huh7.5 cells infected with cell culture grown virus (HCVcc). Silencing of PI4KIIIα further inhibited HCV release into the tissue culture medium. NS5A may recruit PI4KIIIα to the HCV RNA replication complex. These data suggest that PI4KIIIα is an essential host factor that supports HCV proliferation and therefore PI4KIIIα may be a legitimate target for anti-HCV therapy.

Modulation of the Transforming Growth Factor-β Signal Transduction Pathway by Hepatitis C Virus Nonstructural 5A Protein

Transforming growth factor-β (TGF-β) is implicated in the pathogenesis of liver disease. TGF-β is involved both in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis C virus (HCV) infection often leads to cirrhosis and hepatocellular carcinoma. HCV nonstructural 5A (NS5A) protein is a multifunctional protein that modulates cytokine-mediated signal transduction pathways. To elucidate the molecular mechanism of HCV pathogenesis, we examined the effect of NS5A protein on TGF-β-stimulated signaling cascades. We show that NS5A protein inhibited the TGF-β-mediated signaling pathway in hepatoma cell lines as determined by reporter gene assay. To further investigate the role of NS5A, we examined the protein/protein interaction between NS5A and TGF-β signal transducers. Both in vitro and in vivo binding data showed that NS5A protein directly interacted with TGF-β receptor I (TβR-I) in hepatoma cell lines. This interaction was mapped to amino acids 148-238 of NS5A. We also found that NS5A protein co-localized with TβR-I in the cytoplasm of Huh7 cells and inhibited TGF-β-mediated nuclear translocation of Smad2. Furthermore, we demonstrate that NS5A protein abrogated the phosphorylation of Smad2 and the heterodimerization of Smad3 and Smad4. To further explore the relevance to viral infection, we examined the effect of the HCV subgenomic replicon on the TGF-β signaling pathway. We show that the HCV subgenomic replicon also inhibited TGF-β-induced signaling cascades. These results indicate that HCV NS5A modulates TGF-β signaling through interaction with TβR-I and that NS5A may be an important risk factor in HCV-associated liver pathogenesis.

Non-structural 5A protein of hepatitis C virus induces a range of liver pathology in transgenic mice.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). However, the mechanism of HCV pathogenesis is not well understood. Our previous in vitro studies suggested that non‐structural 5A (NS5A) protein may play an important role in liver pathogenesis. To elucidate the mechanism of HCV‐induced liver pathogenesis, we investigated the histopathological changes of liver in transgenic mice harbouring the NS5A gene. We generated transgenic mice harbouring HCV NS5A gene under the control of hepatitis B virus (HBV) enhancer. Pathological changes were analysed by immunohistochemical staining and western blot analysis. Lipid composition and reactive oxygen species (ROS) production in NS5A transgenic mice were analysed. HCV NS5A transgenic mice developed extraordinary steatosis over 6 months old and induced HCC in some mice. NS5A was co‐localized with apolipoprotein A‐I in fatty hepatocytes. In addition, the extraordinarily high levels of ROS, NF‐κB and STAT3 were detected in hepatocytes of NS5A transgenic mice. These data suggest that NS5A, independent of other HCV viral proteins, may play an important role in the development of hepatic pathologies, including steatosis and hepatoceullular carcinoma in transgenic mice. Copyright

Hepatitis C Virus Nonstructural 5B Protein Regulates Tumor Necrosis Factor Alpha Signaling through Effects on Cellular IκB Kinase

ABSTRACT Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNA-dependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-α)-induced NF-κB and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-α-induced NF-κB and JNK activation. In this study, we have demonstrated that TNF-α-induced NF-κB activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2- and IKK-induced NF-κB activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKKα. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKKα, and then TNF-α-mediated IKKα kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-α-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-α signaling pathways and may contribute to HCV pathogenesis.

Peptidyl-Prolyl Isomerase Pin1 Is a Cellular Factor Required for Hepatitis C Virus Propagation

ABSTRACT The life cycle of hepatitis C virus (HCV) is highly dependent on cellular factors. Using small interfering RNA (siRNA) library screening, we identified peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) as a host factor involved in HCV propagation. Here we demonstrated that silencing of Pin1 expression resulted in decreases in HCV replication in both HCV replicon cells and cell culture-grown HCV (HCVcc)-infected cells, whereas overexpression of Pin1 increased HCV replication. Pin1 interacted with both the NS5A and NS5B proteins. However, Pin1 expression was increased only by the NS5B protein. Both the protein binding and isomerase activities of Pin1 were required for HCV replication. Juglone, a natural inhibitor of Pin1, inhibited HCV propagation by inhibiting the interplay between the Pin1 and HCV NS5A/NS5B proteins. These data indicate that Pin1 modulates HCV propagation and may contribute to HCV-induced liver pathogenesis.

The ribonuclease l‐dependent antiviral roles of human 2′,5′‐oligoadenylate synthetase family members against hepatitis C virus

The latent ribonuclease RNase L and the interferon‐inducible 2′,5′‐oligoadenylate synthetase (OAS) have been implicated in the antiviral response against hepatitis C virus (HCV). However, the specific roles of these enzymes against HCV have not been fully elucidated. In this study, a scarce endogenous expression and RNA degrading activity of RNase L in human hepatoma Huh7 cells enabled us to demonstrate the antiviral activity of RNase L against HCV replication through the transient expression of the enzyme. The antiviral potential of specific members of the OAS family was further examined through overexpression and RNA interference approaches. Our data suggested that among the members of the OAS family, OAS1 p46 and OAS3 p100 mediate the RNase l‐dependent antiviral activity against HCV.

Hepatitis C virus core protein potentiates c-Jun N-terminal kinase activation through a signaling complex involving TRADD and TRAF2

The hepatitis C virus (HCV) core protein is a multifunctional viral nucleocapsid protein. Previously, it has been demonstrated that the HCV core protein interacts with the cytoplasmic domain of tumor necrosis factor receptor 1 (TNFR1). Since the TNFR1 is engaged in stimulation of transcriptional factor NF-kappaB and AP-1 through activation of IkappaB kinase and c-Jun N-terminal kinase (JNK, or stress-activated protein kinase), respectively, we have examined whether the interaction between core protein and TNFR1 can modulate JNK. In this study, we demonstrate that the HCV core protein synergistically activates TNFalpha-induced JNK at a core concentration dependent manner in human embryonic kidney (HEK) 293 cells. HCV core-mediated synergism of JNK activation was also detected in stable cells expressing HCV core protein. Furthermore, we demonstrate that HCV core protein does not compete with TNF receptor-associated death domain (TRADD) for its interaction with the death domain of TNFR1. Our in vivo data show that HCV core and TRADD form a ternary complex with TNFR1. These findings suggest that the HCV core protein modulates TNFR1 signaling and may, thus, play a role in chronic infection of HCV patients.