Yun-Sook Lim

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.

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.

Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication

ABSTRACT 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. IMPORTANCE Stearoyl 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.

Modulation of Mitogen-Activated Protein Kinase-Activated Protein Kinase 3 by Hepatitis C Virus Core Protein

ABSTRACT Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ∼9,000 host proteins immobilized in a microarray, approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA and protein levels of MAPKAPK3 were elevated in both HCV subgenomic replicon cells and cell culture-derived HCV (HCVcc)-infected cells. Silencing of MAPKAPK3 expression resulted in decreases in both protein and HCV infectivity levels but not in the intracellular HCV RNA level. We showed that MAPKAPK3 increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation.

Hepatitis delta virus large antigen sensitizes to TNF-α-induced NF-κB signaling

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-κB signaling pathway. In this study, we demonstrated that TNF-α-induced NF-κB transcriptional activation was increased by LHDAg but not by SHDAg in both HEK293 and Huh7 cells. Furthermore, LHDAg promoted TRAF2-induced NF-κB 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-κB activity. We further showed that only LHDAg but not SHDAg increased the TNF-α-mediated nuclear translocation of p65. This was accomplished by activation of IκBα degradation by LHDAg. Finally, we demonstrated that LHDAg augmented the COX-2 expression level in Huh7 cells. These data suggest that LHDAg modulates NF-κB signaling pathway and may contribute to HDV pathogenesis.

Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry

ABSTRACT 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 ∼9,000 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. IMPORTANCE Pim1 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 HCV-infected 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.

Saponin Inhibits Hepatitis C Virus Propagation by Up-regulating Suppressor of Cytokine Signaling 2

Saponins are a group of naturally occurring plant glycosides which possess a wide range of pharmacological properties, including anti-tumorigenic and antiviral activities. To investigate whether saponin has anti-hepatitis C virus (HCV) activity, we examined the effect of saponin on HCV replication. HCV replication was efficiently inhibited at a concentration of 10 µg/ml of saponin in cell culture grown HCV (HCVcc)-infected cells. Inhibitory effect of saponin on HCV replication was verified by quantitative real-time PCR, reporter assay, and immunoblot analysis. In addition, saponin potentiated IFN-α-induced anti-HCV activity. Moreover, saponin exerted antiviral activity even in IFN-α resistant mutant HCVcc-infected cells. To investigate how cellular genes were regulated by saponin, we performed microarray analysis using HCVcc-infected cells. We demonstrated that suppressor of cytokine signaling 2 (SOCS2) protein level was distinctively increased by saponin, which in turn resulted in inhibition of HCV replication. We further showed that silencing of SOCS2 resurrected HCV replication and overexpression of SOCS2 suppressed HCV replication. These data imply that saponin inhibits HCV replication via SOCS2 signaling pathway. These findings suggest that saponin may be a potent therapeutic agent for HCV patients.

SUMOylation of nonstructural 5A protein regulates hepatitis C virus replication

Viruses exploit cellular SUMOylation machinery to favour their own propagation. We show that NS5A is a target protein of small ubiquitin‐like modifier (SUMO) and is SUMOylated at lysine residue 348. We demonstrated that SUMOylation increased protein stability of NS5A by inhibiting ubiquitylation, and SUMOylation was also required for protein interaction with NS5B. These data imply that SUMO modification may contribute to HCV replication. Indeed, silencing of UBC9 impaired HCV replication in Jc1‐infected cells, and HCV replication level was also significantly reduced in SUMO‐defective subgenomic replicon cells. Taken together, these data indicate that HCV replication is regulated by SUMO modification of NS5A protein. We provide evidence for the first time that HCV exploits host cellular SUMO modification system to favour its own replication.

Farnesyl‐diphosphate farnesyltransferase 1 regulates hepatitis C virus propagation

To identify the novel genes involved in lipid metabolism and lipid droplet formation that may play important roles in Hepatitis C virus (HCV) propagation, we have screened the small interfering RNA library using cell culture derived HCV (HCVcc)‐infected cells. We selected and characterized the gene encoding farnesyl‐diphosphate farnesyltransferase 1 (FDFT1). siRNA‐mediated knockdown of FDFT1 impaired HCV replication in both subgenomic replicon and HCVcc infected cells. Moreover, YM‐53601, an inhibitor of FDFT1 enzyme activity, abrogated HCV propagation. HCV infection increased FDFT1 protein level but not FDFT1 mRNA level. These results suggest that HCV may modulate FDFT1 protein level to facilitate its own propagation.

Nonstructural 5A protein of hepatitis C virus regulates heat shock protein 72 for its own propagation

Summary.  We identified heat shock protein 72 (Hsp72) as a host factor that was differentially expressed in cells expressing nonstructural 5A (NS5A) protein. To investigate how NS5A modulates Hsp72 in hepatitis C virus (HCV) life cycle, we examined the role of Hsp72 in HCV replication and virus production. NS5A specifically interacted with Hsp72. Both Hsp72 and nuclear factor of activated T cells 5 (NFAT5) levels were increased in cells expressing NS5A protein. Treatments of N‐acetylcysteine and glutathione markedly reduced protein levels of both NFAT5 and Hsp72. Knockdown of NFAT5 resulted in decrease in Hsp72 level in cells expressing NS5A. Importantly, silencing of Hsp72 expression resulted in decrease in both RNA replication and virus production in HCV‐infected cells. These data indicate that NS5A modulates Hsp72 via NFAT5 and reactive oxygen species activation for HCV propagation.