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

Min

et al. 2015

Self Cite

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.

Section: Discussionmentioning

confidence: 99%

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

Min

et al. 2015

Self Cite

“…Total amounts of DNA were adjusted by adding an empty vector. At 48 h after transfection, cells were harvested and an immunoprecipitation assay was performed as described previously (15). For subgenomic replicon cells and Jc1-infected Huh7.5 cells, Flag-tagged SCD1 was transfected and harvested at 48 h after transfection using hypotonic buffer (10 mM Tris-HCl, pH 7.8, 10 mM NaCl).…”

Section: Methodsmentioning

confidence: 99%

“…PCR products were inserted into the HindIII and EcoRI sites of the plasmid pCMV10-3x Flag (Sigma-Aldrich) or pGFP-C1. pEF6B Myc-tagged HCV core, NS3, NS4B, NS5A, and NS5B were described previously (15). siRNA-resistant mutant SCD1 (pCMV10-SCD1-SR) was constructed by introducing five silent mutations at the siRNA binding site with a site-directed mutagenesis kit (Enzynomics).…”

Section: Methodsmentioning

confidence: 99%

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

Nguyen

Lim

Pham

et al. 2014

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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...

“…In addition, various cellular factors involved in viral replication have been identified through interaction with NS5A (44). Thus, we coexpressed GFP-tagged vinexin ␤ and red fluorescent protein (RFP)-fused binding partners of NS5A such as annexin A2, CK1␣, CK2␣, CyPA, FBXL2, FKBP8, hB-ind1, Hsp72, Pin1, PSTPIP2, VAP-A, and VAP-B in Huh7-Lunet cells (6,13,(49)(50)(51)(52)(53)(54)(55)(56)(57). The results showed that host kinase RFP-CK1␣ was obviously colocalized with GFP-vinexin ␤, but RFP-VAP-A and RFP-CK2␣ were not (6,58) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation

Xiong

Yang

Wang

et al. 2015

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is essential for HCV genome replication and virion production and is involved in the regulation of multiple host signaling pathways. As a proline-rich protein, NS5A is capable of interacting with various host proteins containing Src homology 3 (SH3) domains. Previous studies have suggested that vinexin, a member of the sorbin homology (SoHo) adaptor family, might be a potential binding partner of NS5A by yeast two-hybrid screening. However, firm evidence for this interaction is lacking, and the significance of vinexin in the HCV life cycle remains unclear. In this study, we demonstrated that endogenously and exogenously expressed vinexin ␤ coimmunoprecipitated with NS5A derived from different HCV genotypes. Two residues, tryptophan (W307) and tyrosine (Y325), in the third SH3 domain of vinexin ␤ and conserved Pro-X-X-Pro-X-Arg motifs at the C terminus of NS5A were indispensable for the vinexin-NS5A interaction. Furthermore, downregulation of endogenous vinexin ␤ significantly suppressed NS5A hyperphosphorylation and decreased HCV replication, which could be rescued by expressing a vinexin ␤ short hairpin RNA-resistant mutant. We also found that vinexin ␤ modulated the hyperphosphorylation of NS5A in a casein kinase 1␣-dependent on manner. Taken together, our findings suggest that vinexin ␤ modulates NS5A phosphorylation via its interaction with NS5A, thereby regulating HCV replication, implicating vinexin ␤ in the viral life cycle. IMPORTANCEHepatitis C virus (HCV) nonstructural protein NS5A is a phosphoprotein, and its phosphorylation states are usually modulated by host kinases and other viral nonstructural elements. Additionally, cellular factors containing Src homology 3 (SH3) domains have been reported to interact with proline-rich regions of NS5A. However, it is unclear whether there are any relationships between NS5A phosphorylation and the NS5A-SH3 interaction, and little is known about the significance of this interaction in the HCV life cycle. In this work, we demonstrate that vinexin ␤ modulates NS5A hyperphosphorylation through the NS5A-vinexin ␤ interaction. Hyperphosphorylated NS5A induced by vinexin ␤ is casein kinase 1␣ dependent and is also crucial for HCV propagation. Overall, our findings not only elucidate the relationships between NS5A phosphorylation and the NS5A-SH3 interaction but also shed new mechanistic insight on Flaviviridae NS5A (NS5) phosphorylation. We believe that our results may afford the potential to offer an antiviral therapeutic strategy. H epatitis C virus (HCV) infection is a global health disease and is a major cause of chronic liver disease leading to hepatic fibrosis, liver cirrhosis, and hepatic carcinoma. No protective vaccine is available. Some directly acting antiviral agents combining pegylated interferon and ribavirin show therapeutic promise for chronic hepatitis C. However, the mechanisms of drug action, the issues of interferon-free therapy, drug resistance, and broad treatment of all HCV genotypes...