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 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.

Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

“…We recently performed a protein microarray assay using the NS5A protein as a probe to identify cellular proteins interacting with NS5A (22). Statistically significant proteins were determined as NS5A interactors.…”

“…To identify host proteins interacting with NS5A, we recently employed protein array screening and identified approximately 90 human candidates as HCV NS5A interactors (22). Since sorcin interacts with the VP1 protein of Huh7.5 cells were transiently transfected with increasing amounts of V5-tagged sorcin expression plasmid together with pRL-HL dual luciferase and a pCH110 ␤-galactosidase plasmid.…”

“…Antibodies were purchased from the following sources: sorcin antibodies were from Santa Cruz and Abcam, c-Myc antibodies were from Santa Cruz, ␤-actin antibodies were from Sigma-Aldrich, and V5 antibodies were from Invitrogen; HCV core, NS3, and NS5A antibodies have been described elsewhere (22). BI2536, a PLK1 inhibitor, was purchased from Selleck Chem.…”

“…For a dual-luciferase reporter assay, Huh7.5 cells were cotransfected with a pRL-HL plasmid containing the Renilla luciferase gene under the cytomegalovirus (CMV) promoter and the firefly luciferase gene under the control of the HCV internal ribosome entry site (IRES) and the plasmid indicated in the figures together with the pCH110 reference plasmid. At 48 h after transfection, cells were harvested, and then luciferase assays were performed as we described previously (22).…”

“…We recently performed protein microarray analysis to identify host factors interacting with HCV NS5A (22). In the present study, we selected sorcin for further characterization.…”