Elevated levels of oncogenic protein kinase Pim-1 induce the p53 pathway in cultured cells and correlate with increased Mdm2 in mantle cell lymphoma. was increased by NS5A protein and this increase was mediated by protein interplay. Small Bifenazate 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 Bifenazate 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. INTRODUCTION Hepatitis C virus (HCV) is a major etiological agent of chronic liver disease, including cirrhosis and hepatocellular carcinoma (1). Approximately 170 million people are chronically infected with HCV, and HCV-related disease leads to 350,000 deaths annually (2). Although recent development of direct-acting antivirals (DAAs) displayed significant progress in HCV treatment regimens, there are still many issues, including unaffordable high cost of drugs, genotypic efficacy, and occasional occurrence of resistance-associated variants. HCV is an enveloped virus with a positive-sense, single-stranded RNA that belongs to the genus within the family (3). The 9.6-kb HCV genome encodes a single polyprotein of 3,010 amino acids, which is sequentially processed into 3 structural proteins (core, E1, and E2) and 7 nonstructural Bifenazate proteins (p7 and NS2 to NS5B) (1, 2). Nonstructural 5A (NS5A) is a multifunctional protein consisting of 447 amino acid residues. NS5A protein exists in two different sizes of polypeptide (p56 and p58), which is phosphorylated mainly at serine residues by cellular kinase (3). NS5A protein interacts with many cellular and viral Rabbit Polyclonal to Smad1 proteins and regulates viral replication and host cellular signaling pathways (4, 5). We have previously reported that NS5A modulates tumor necrosis factor alpha (TNF-) signaling of the host cells through the interaction with TRAF2 (6) and also regulates TGF- signaling (7), which are implicated in HCV-associated liver pathogenesis. In addition, we showed that NS5A modulated -catenin signaling that might play a crucial role in HCV pathogenesis (8). More recently, we reported that NS5A interacted with cellular Pin1 (9) and PI4KIII (10), and regulated HCV replication. All these data firmly support the idea that NS5A not only plays an important role in HCV replication but also contributes to HCV-mediated liver pathogenesis. The provirus integration site for Moloney murine leukemia virus (Pim1) was first identified as an activated gene in Molony murine leukemia virus-induced T cell lymphoma (11). Pim1 belongs to an oncogenic serine/threonine kinase family with two other members, Pim2 and Pim3. Pim1 shares sequence homologies of 71% with Pim2 and 61% with Pim3. Pim1 is a proto-oncogene whose activation promotes the development of cancer in animals (12, 13). Pim kinases are involved in various cellular processes, including cell cycle regulation, proliferation, apoptosis, and signal transduction pathways (11). Overexpression of Pim contributes to malignant transformation and tumorigenesis (14, 15) as well as the expression degrees of Pim protein are connected with their real activities. Indeed, it’s been previously reported that Pim kinases are upregulated in solid tumors and hepatoma cells (16,C18). Using proteins microarray analysis, we’ve identified 90 NS5A-interacting mobile proteins approximately. Right here we present that NS5A interacts with Pim1 physically. Proteins connections was verified by both coimmunoprecipitation and pulldown assays. Moreover, NS5A elevated proteins balance of Pim1 through downregulation from the polyubiquitination procedure. Silencing of Pim kinases abrogated HCV propagation. This is further verified with a Pim kinase inhibitor. We further demonstrated that Pim kinases get excited about the entry stage of HCV an infection. These data claim that Pim proteins may be the best focus on for anti-HCV therapy. Strategies and Components Plasmids and DNA transfection. Total RNAs had been isolated from Huh7 cells through the use of RiboEx (GeneAll), and full-length Pim1, Pim2, Pim3, and Poor had been amplified from cDNA synthesized with a cDNA synthesis package (Toyobo) based on the manufacturer’s guidelines. PCR products had been inserted in to the matching enzyme sites from the plasmid pCMV10-3x Flag (Sigma-Aldrich). Pim1 was subcloned into either the plasmid Bifenazate pGEX-4T-1 (Amersham Biosciences Stomach, Uppsala, Sweden) or pGFP-C1. pEF6B.
