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Hepatitis C virus (HCV)2 is a single-stranded positive-sense RNA virus capable of establishing a chronic infection in 70 -80% of HCV-infected patients. Approximately 3% of the population of the world are chronically infected with HCV. Over 30% of them will develop cirrhosis within 20 years, which subsequently may lead to hepatocellular carcinoma (1-3). A vaccine is not available and the optimal current therapy for patients infected with HCV is a combination of the pegylated interferon (IFN) and ribavirin. However, the therapy with these agents is prolonged, costly, and associated with a high rate of side effects. Furthermore, the sustained virologic response rate to the combination therapy varies with different genotypes of HCV. Although small molecule inhibitors of viral protease and RNA-dependent RNA polymerase are in clinical development, the error prone nature of the viral RNA polymerase leads to rapid emergence of viral-resistant mutations to these therapeutic candidates. Therefore, a new approach to control HCV proliferation could be identifying the host factors involved in the HCV life cycle. HCV relies heavily on host proteins for all steps of its life cycle, including viral entry, uncoating, replication, assembly, and virion release. Therefore, any step that can interrupt the HCV life cycle will be a putative target for HCV therapy.Nonstructural 5A (NS5A) protein is a multifunctional phosphoprotein consisting of 447 amino acids residues. NS5A protein is localized in the cytoplasm and forms part of the HCV RNA replication complex (4). NS5A has been shown to interact with many host factors, including TRAF2, hVAP-33, PKR, and Grb2, to regulate viral replication and cellular signaling pathways (5-8). A growing body of evidence indicates that NS5A may play important roles in the HCV-induced liver pathogenesis. We have shown that NS5A modulated TNF␣ signaling of the host cells through the NS5A-TRAF2 interaction (5). In addition, we demonstrated that NS5A induced steatosis and hepatocellular carcinoma in transgenic mice (9). Recently, we also showed that NS5A modulated ␤-catenin signaling that might play an important role in HCV pathogenesis (10).Phosphatidylinositol 4-kinase type III␣ (PI4KIII␣) is a lipid kinase that is encoded by the PI4KCA gene in human (11). PI4KIII␣ phosphorylates phosphatidylinositol (PtdIns) to phosphatidylinositol 4-P, which can be further phosphorylated by PIP5 kinases to phosphatidylinositol (4,5)-P 2 . PI4KIII␣ is localized primarily to the endoplasmic reticulum and regulates endoplasmic reticulum exit sites (12, 13). Recently, PI4KIII␣ has been identified as a cellular factor involved in the HCV life cycle using siRNA library screening (14 -19). However, how PI4KIII␣ regulates HCV proliferation is not clearly understood. In this study, we identified PI4KIII␣ as a binding partner for the NS5A protein. Silencing of PI4KIII␣ significantly reduced HCV replication and virion release in HCV-infected cells. These data suggest that HCV may modulate cellular PI4KIII␣ for its own RNA replic...

The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ϳ9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry. IMPORTANCEPim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCVinfected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell entry factor but also as a new anti-HCV therapeutic target.

The hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet formation using cell culture-grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl coenzyme A (CoA) desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1 is associated with HCV replication complex. Moreover, SCD1 was fractionated and enriched with HCV nonstructural proteins at detergent-resistant membrane. Electron microscopy data showed that SCD1 is required for NS4B-mediated intracellular membrane rearrangement. These data further support the idea that SCD1 is associated with HCV replication complex and that its products may contribute to the proper formation and maintenance of membranous web structures in HCV replication complex. Collectively, these data suggest that manipulation of SCD1 activity may represent a novel host-targeted antiviral strategy for the treatment of HCV infection. IMPORTANCEStearoyl coenzyme A (CoA) desaturase 1 (SCD1), a liver-specific enzyme, regulates hepatitis C virus (HCV) replication through its enzyme activity. HCV nonstructural proteins are associated with SCD1 at detergent-resistant membranes, and SCD1 is enriched on the lipid raft by HCV infection. Therein, SCD1 supports NS4B-mediated membrane rearrangement to provide a suitable microenvironment for HCV replication. We demonstrated that either genetic or chemical knockdown of SCD1 abrogated HCV replication in both replicon cells and HCV-infected cells. These findings provide novel mechanistic insights into the roles of SCD1 in HCV replication. Hepatitis C virus (HCV) is an enveloped virus with a positivesense, single-stranded RNA virus that belongs to the genus Hepacivirus in the family Flaviviridae (1). Approximately 170 million people are chronically infected with HCV worldwide. Three million people are newly infected with HCV annually, and more than 350,000 individuals die from HCV-related liver diseases every year (1, 2). Current standard therapy elicits some side effects and results in a sustained virological response in only certain genotypes of HCV patients. Recently, the U.S. Food and Drug Administration approved various direct-acting antivirals (DAAs), including boceprevir, telaprevir, sofosbuvir, and simeprevir, for triple therapy in combination with pegylated interferon and ribavirin for patients with certain genotypes. Nevertheless, these new DAAs also have had some lim...

Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV viral proteins, the small hepatitis delta antigen (SHDAg) and the large hepatitis delta antigen (LHDAg), on NF-kappaB signaling pathway. In this study, we demonstrated that TNF-alpha-induced NF-kappaB transcriptional activation was increased by LHDAg but not by SHDAg in both HEK293 and Huh7 cells. Furthermore, LHDAg promoted TRAF2-induced NF-kappaB activation. Using coimmunoprecipitation assays, we demonstrated that both SHDAg and LHDAg interacted with TRAF2 protein. We showed that isoprenylation of LHDAg was not required for the increase of NF-kappaB activity. We further showed that only LHDAg but not SHDAg increased the TNF-alpha-mediated nuclear translocation of p65. This was accomplished by activation of IkappaBalpha degradation by LHDAg. Finally, we demonstrated that LHDAg augmented the COX-2 expression level in Huh7 cells. These data suggest that LHDAg modulates NF-kappaB signaling pathway and may contribute to HDV pathogenesis.